Antibodies specific for ungulate PrP

ABSTRACT

The present invention provides antibodies that specifically bind with a high degree of binding affinity to a native ungulate PrP C  and/or a denatured ungulate PrP Sc , but not to a native ungulate PrP Sc . Preferred antibodies find native bovine PrP C  and treated PrP Sc  but not native bovine PrP Sc  and can be used in an assay to determine if a sample is infected with infectious prions, i.e. PrP Sc .

CROSS-REFERENCE

This application is a continuation-in-part application of Ser. No.09/627,218, filed Jul. 27, 2000, now issued U.S. Pat. No. 6,537,548 onMar. 25, 2003, which is incorporated herein by reference in its entiretyand to which application we claim priority under 35 USC §120.

GOVERNMENT RIGHTS

The United States Government may have certain rights in this applicationpursuant to Contract No. HL 63817 awarded by the National Institutes ofHealth.

FIELD OF THE INVENTION

This invention relates to antibodies, methods for obtaining antibodiesand assays for using such antibodies. More specifically, the inventionrelates to ungulate PrP antibodies methods of obtaining antibodies whichspecifically bind to naturally occurring forms of PrP from ungulates.

BACKGROUND OF THE INVENTION

Prions are infectious pathogens that cause central nervous systemspongiform encephalopathies in humans and animals. Prions are distinctfrom bacteria, viruses and viroids. The predominant hypothesis atpresent is that no nucleic acid component is necessary for infectivityof prion protein. Further, a prion which infects one species of animal(e.g., a human) will not readily infect another (e.g., a mouse).

A major step in the study of prions and the diseases that they cause wasthe discovery and purification of a protein designated prion protein(“PrP”) (Bolton et al. (1982), Science 218:1309–11; Prusiner et al.(1982), Biochemistry 21:6942–50; McKinley et al.(1983), Cell 35:57–62).Complete prion protein-encoding genes have since been cloned, sequencedand expressed in transgenic animals. PrP^(C) is encoded by a single-copyhost gene (Basler et al.(1986), Cell 46:417–28) and is normally found atthe outer surface of neurons. A leading hypothesis is that priondiseases result from conversion of PrP^(C) into a modified form calledPrP^(Sc).

It appears that PrP^(Sc) is necessary for both the transmission andpathogenesis of the transmissible neurodegenerative diseases of animalsand humans. See Prusiner, S. B.(1991), Science 252:1515–1522. The mostcommon prion diseases of animals are scrapie of sheep and goats, andbovine spongiform encephalopathy (BSE) of cattle (Wilesmith, J. andWells (1991), Microbiol. Immunol. 172:21–38). Four prion diseases ofhumans have been identified: (1) kuru, (2) Creutzfeldt-Jakob Disease(CJD), (3) Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatalfamilial insomnia (FFI) (Gajdusek, D. C., (1977) Science 197:943–960;Medori et al.(1992), N. Engl. J. Med. 326:444–449). The presentation ofhuman prion diseases as sporadic, genetic and infectious illnessesinitially posed a conundrum which has been explained by the cellulargenetic origin of PrP.

BSE is also a major socioeconomic problem, particularly in Britain. Morethan 175,000 cattle, primarily dairy cows, have died of BSE during thepast decades. Tests conducted by the British government on cattle killedover a 30 month period suggest that about 0.3% of the 749,631 tested, or2249 cattle may have had BSE even though they did not display anyoutward symptoms. On Mar. 27, 1996, the European Union (EU) placed a banon the export of British bovine products, including: live bovineanimals, their semen and embryos; meat of bovine animals slaughtered inUK; products obtained from bovine animals slaughtered in UK which areliable to enter the animal feed or human food chain; materials destinedfor use in medicinal products, cosmetics or pharmaceutical products; andmammalian derived meat and bone-meal. This ban has cost the Britishfarming industry more than 1.5 billion pounds since it was imposed, andleft many farmers bankrupt. Although the ban was lifted Aug. 1, 1999,both France and Germany still ban the import of British bovine products,contrary to the EU ruling.

The importance of detecting BSE has been heightened by the possibilitythat bovine prions have been transmitted to humans who developed newvariant Creutzfeldt-Jakob disease (nvCJD) (G. Chazot et al.(1996),Lancet 347:1181; R. G. Will et al. (1996), Lancet 347:921–925). Earlierstudies had shown that the N-terminus of PrP^(Sc) could be truncatedwithout loss of scrapie infectivity (S. B. Prusiner et al.(1982),Biochemistry 21:6942–6950; S. B. Prusiner et al. (1984), Cell38:127–134) and correspondingly, the truncation of the N-terminus ofPrP^(Sc) still allowed its conversion into PrP^(Sc) (M. Rogers etal.(1993), Proc. Natl. Acad. Sci. USA 90:3182–3186). The ability oftransmission of nvCJD from cattle to humans has been confirmed throughin vivo testing, suggesting that the December 20 issue of Proceedings ofNational Academy of Sciences undermining the comforting presumption thatthe documented “species barrier” is relevant to this new strain (M. R.Scott et al. (1999), Proc. Natl. Acad. Sci. USA 96:15137–15142).

The presence of PrP^(Sc) in tissues of humans or animals is indicativeof prion infection. PrP^(Sc) is the invariant component of prioninfection and is the only disease-specific diagnostic marker that can bereadily detected by immunoassay in the brains of clinically ill animalsand humans Meyer et al. (1986), Proc. Natl. Acad. Sci. USA, 83:3693–7;Serban et al. (1990), Neurology, 40:110–117; Taraboulos et al. (1992),Proc. Natl. Acad. Sci. USA. 89:7620–7624; Grathwohl, K. U. D., M.Horiuchi et al. (1997), Virol. Methods 64:205–216. Unfortunately,PrP^(Sc) assays are positive only when the prion titer is high, whiledetection of low levels of PrP^(Sc) has been problematic. It has alsoproven difficult to measure low levels of PrP^(Sc) in the presence ofhigh levels of PrP^(C).

Given the enormity of the potential effect of BSE on the world widecattle population and the affected cattle population in Great Britain,there is a great need for a method of assessing bovine infection withBSE to protect the cattle populations. Given the potential health riskto the human population, more sensitive methods for detection of bovineprions are urgently needed.

SUMMARY OF THE INVENTION

The present invention provides antibodies that will specifically bindwith a high degree of affinity to a native ungulate PrP^(C) and/or adenatured ungulate PrP^(Sc), but not to a native ungulate PrP^(Sc). Theantibodies are also highly specific, i.e. do not bind to other proteins.The antibodies are useful in numerous applications, and particularly fordetermining prion infection in ungulates. The antibodies arecharacterized by one or more of the following features (1) an ability tobind to native PrP^(C) and denatured PrP^(Sc), but not native PrP^(Sc),with specificity, (2) an ability to bind to PrP^(C) or denaturedPrP^(Sc) in situ i.e., will only bind to PrP^(Sc) in a cell culture orin vivo if the prion protein has been treated (e.g. denatured).Preferred antibodies are further characterized by an ability to (4) bindto a PrP^(C) protein of only a specific species of mammals e.g., bind tobovine PrP^(C) and not to PrP^(C) of other mammals.

An important object is to provide antibodies which bind to a native formof ungulate PrP^(C).

Another object is to provide antibodies which specifically bind toepitopes of PrP^(C) of a specific species of animal (e.g. bovinePrP^(C)) and not to the PrP^(C) of other species of animals (e.g. mousePrP^(C)).

Still another object is to provide specific methodology to allow othersto generate a wide range of specific antibodies characterized by theirability to bind one or more types of PrP^(C) proteins from one or morespecies of ungulates.

Another object of the invention is to provide an assay for the detectionof PrP^(Sc) in an ungulate using the antibodies of the invention.

An advantage of the invention is that it provides a fast, efficient costeffective assay for detecting the presence of PrP^(Sc) in an ungulatesample.

A specific advantage is that the assay can be used as a screen for thepresence of prions (i.e., PrP^(Sc)) in products such as pharmaceuticals(derived from natural sources) food, cosmetics or any material whichmight contain such prions and thereby provide further assurances as tothe safety of such products.

Another advantage is that the antibodies can be used with a compoundwhich denatures PrP^(Sc) thereby providing for a means ofdifferentiating levels of PrP^(C) and PrP^(C)+PrP^(Sc) in a sample.

A feature of the invention is that it uses phage display libraries inthe creation of the antibodies.

Another feature of the invention is that the phage are geneticallyengineered to express a specific binding protein of an antibody on theirsurface.

An aspect of the invention is to provide a therapeutic antibody whichprevents or treats prion disease in ungulates and specifically in cows.

Another aspect of the invention is to provide a means for certifyingcertain products as being prion free.

These and other aspects, objects, advantages, and features of theinvention will become apparent to those persons skilled in the art uponreading the details of the chimeric gene, assay method, and transgenicmouse as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of the steps of the conformation-dependantimmunoassay (CDI) uses anti-bovine antibodies described and disclosedhere.

FIG. 2A shows the amino acid sequence of mouse PrP (SEQ ID NO.: 10) andFIG. 2B shows the amino acid sequence of bovine PrP (SEQ ID NO.: 11).

FIG. 3 illustrates the sensitivity of conformation-dependent immunoassay(CDI) in the detection of chimeric MBo2M PrP by Eu-(HuM)Fab P andEu-(HuM)Fab S.

FIGS. 4 and 5 illustrate the sensitivity of CDI for detection of BovinePrP^(Sc) in the brain homogenates of BSE-infected Tg(BoPrP) mice usingEu-(HuM)Fab P.

FIGS. 6 and 7 illustrate the sensitivity of CDI for detection of bovinePrP^(Sc) in the brain homogenates of BSE-infected British cows usingEu-(HuM) Fab P.

FIG. 8 illustrates the concentration of PrP 27–30 plotted againstdenatured/native ratio determined by CDI in 32 British cows infected byBSE and 12 noninfected U.S. controls.

FIGS. 9 and 10 show the results of CDI testing for PrP^(Sc) in chronicwasting diseases (CWD)-infected mule deer, elk, white-tail deer, andnormal controls.

FIGS. 11 and 12 illustrate the sensitivity of CDI for detection of deerPrP^(Sc) in the frontal cortex of CWD-infected deer using Eu-(HuM) FabP. Dynamic range of the detection of deer PrP^(Sc) is ≧100,000-fold.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Before the present antibodies, assays and methods for producing andusing such are disclosed and described, it is to be understood that thisinvention is not limited to particular antibodies, assays or method assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

Definitions

The terms “PrP protein”, “PrP” and the like are used interchangeablyherein and shall mean both the infectious particle form PrP^(Sc) knownto cause diseases (spongiform encephalopathies) in humans and animalsand the non-infectious form PrP^(C) which, under appropriate conditionsis converted to the infectious PrP^(Sc) form.

The terms “prion”, “prion protein” and “PrP^(Sc) protein” and the likeused interchangeably herein to refer to the infectious PrP^(Sc) form ofa PrP protein and is a contraction of the words “protein” and“infection” and the particles are comprised largely if not exclusivelyof PrP^(Sc) molecules encoded by a PrP gene. Prions are distinct frombacteria, viruses and viroids. Known prions include those which infectanimals to cause scrapie, a transmissible, degenerative disease of thenervous system of sheep and goats as well as bovine spongiformencephalopathies (BSE) or mad cow disease and chronic wasting disease(CWD) of deer and elk. Four prion diseases known to affect humans are(1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familialinsomnia (FFI). As used herein prion includes all forms of prionscausing all or any of these diseases or others in any animals used—andin particular in humans and in domesticated farm animals.

The term “PrP gene” is used herein to describe genetic material whichexpresses PrP^(C) proteins, including proteins having polymorphisms andmutations such as those listed herein under the subheading “PathogenicMutations and Polymorphisms.” The term “PrP gene” refers generally toany gene of any species which encodes any form of a prion protein. Somecommonly known PrP sequences are described in Gabriel et al. (1992),Proc. Natl. Acad. Sci. USA 89:9097–9101, which is incorporated herein byreference to disclose and describe such sequences. The PrP gene can befrom any animal including the “host” and “test” animals described hereinand any and all polymorphisms and mutations thereof, it being recognizedthat the terms include other such PrP genes that are yet to bediscovered. The protein expressed by such a gene can assume either aPrP^(C) (non-disease) of PrP^(Sc) (disease) form.

The terms “standardized prion preparation”, “prion preparation”,“preparation” and the like are used interchangeably herein to describe acomposition containing prions (PrP^(Sc)) which composition is obtainedfrom brain tissue of mammals which contain substantially the samegenetic material as relates to prions, e.g., brain tissue from a set ofmammals which exhibit signs of prion disease which mammals (1) include atransgene as described herein; (2) have an ablated endogenous prionprotein gene; (3) have a high copy number of prion protein gene from agenetically diverse species; or (4) are hybrids with an ablatedendogenous prion protein gene and a prion protein gene from agenetically diverse species. The mammals from which standardized prionpreparations are obtained exhibit clinical signs of CNS dysfunction as aresult of inoculation with prions and/or due to developing the diseasedue to their genetically modified make up, e.g., high copy number ofprion protein genes.

The term “artificial PrP gene” is used herein to encompass the term“chimeric PrP gene” as well as other recombinantly constructed geneswhich when included in the genome of a host animal (e.g., a mouse) willrender the mammal susceptible to infection from prions which naturallyonly infect a genetically diverse test mammal, e.g., human, bovine orovine. In general, an artificial gene will include the codon sequence ofthe PrP gene of the mammal being genetically altered with one or more(but not all, and generally less than 40) codons of the natural sequencebeing replaced with a different codon—preferably a corresponding codonof a genetically diverse mammal (such as a cow). The genetically alteredmammal can be used to assay samples for prions which only infect thegenetically diverse mammal. Examples of artificial genes are mouse PrPgenes having one or more different replacement codons from cows, sheepand the like replacing mouse codons at the same relative position, withthe proviso that not all the mouse codons are replaced with differinghuman, cow or sheep codons. Artificial PrP genes can include not onlycodons of genetically diverse animals but may include codons and codonsequences not associated with any native PrP gene but which, wheninserted into an animal, render the animal susceptible to infection withprions which would normally only infect a genetically diverse animal.

The terms “chimeric gene”, “chimeric PrP gene”, “chimeric prion proteingene” and the like are used interchangeably herein to mean anartificially constructed gene containing the codons of a host animal,such as a mouse, with one or more of the codons being replaced withcorresponding codons from a genetically diverse test animal, such as acow or sheep. In one specific example the chimeric gene is comprised ofthe starting and terminating sequence (i.e., N- and C- terminal codons)of a PrP gene of a mammal of a host species (e.g. a mouse) and alsocontaining a nucleotide sequence of a corresponding portion of a PrPgene of a test mammal of a second species (e.g. a cow). A chimeric genewill, when inserted into the genome of a mammal of the host species,render the mammal susceptible to infection with prions which normallyinfect only mammals of the second species. The preferred chimeric genedisclosed herein is MBo2M which contains the starting and terminatingsequence of a mouse PrP gene and a non-terminal sequence region replacedwith a corresponding bovine sequence. The bovine sequence differs from amouse PrP gene in a manner such that the protein expressed therebydiffers at nine residues (see FIG. 2B). MBo2M PrP was constructed asdescribed previously for similar chimeric PrP transgenes (Scott, M., D.Groth et al. (1993), Cell 73: 979–988) and resulting in eight bovinesubstitutions in MoPrP (position numbers correspond to HuPrP sequence):97, 109, 138, 143, 145, 155, 184 and 186.

The term “genetic material related to prions” is intended to cover anygenetic material which effects the ability of an animal to becomeinfected with prions. Thus, the term encompasses any “PrP gene”,“artificial PrP gene”, “chimeric PrP gene” or “ablated PrP gene” whichterms are defined herein as well as modification of such which effectthe ability of an animal to become infected with prions. Standardizedprion preparations are produced using animals which all havesubstantially the same genetic material related to prions so that all ofthe animals will become infected with the same type of prions and willexhibit signs of infection at about the same time.

The terms “host animal” and “host mammal” are used to describe animalswhich will have their genome genetically and artificially manipulated soas to include genetic material which is not naturally present within theanimal. For example, host animals include mice, hamsters and rats whichhave their PrP gene ablated, i.e., rendered inoperative. The host isinoculated with prion proteins to generate antibodies, and the cellsproducing the antibodies can be a source of genetic material for makinga phage library. Other host animals can have a natural (PrP) gene or onewhich is altered by the insertion of an artificial gene or by theinsertion of a native PrP gene of a genetically diverse test animal.

The terms “test animal” and “test mammal” are used to describe theanimal which is genetically diverse from the host animal in terms ofdifferences between the PrP gene of the host animal and the PrP gene ofthe test animal. The test animal may be any animal for which one wishesto run an assay test to determine whether a given sample contains prionswith the ability to infect test animal. For example, the test animal maybe any ungulate or mammal infected with a variant ungulate prion,including human, cow, sheep, pig, horse, cat, dog or chicken, and onemay wish to determine whether a particular sample includes prions whichwould normally infect only the test animal.

The terms “genetically diverse animal” and “genetically diverse mammal”are used herein to describe an animal which includes a native PrP codonsequence of the host animal differing from the genetically diverse testanimal by 17 or more codons, preferably 20 or more codons, and mostpreferably 28–40 codons. Thus, a mouse PrP gene is genetically diversewith respect to the PrP gene of a cow or sheep, but is not geneticallydiverse with respect to the PrP gene of a hamster.

The terms “ablated PrP protein gene”, “disrupted PrP gene”, and the likeare used interchangeably herein to mean an endogenous PrP gene which hasbeen altered (e.g., added and/or removed nucleotides) in a manner so asto render the gene inoperative. Examples of non-functional PrP genes andmethods of making such are disclosed in Büieler, H., et al. (1992),Nature 356, 577–582 and Weissman (WO 93/10227). The methodology forablating a gene is taught in Capecchi (1987), Cell 51:503–512, all ofwhich are incorporated herein by reference. Preferably both alleles ofthe genes are disrupted.

The terms “hybrid animal”, “transgenic hybrid animal” and the like areused interchangeably herein to mean an animal obtained from thecross-breeding of a first animal having an ablated endogenous prionprotein gene with a second animal which includes either (1) a chimericgene or artificial PrP gene or (2) a PrP gene from a genetically diverseanimal. For example a hybrid mouse is obtained by cross-breeding a mousewith an ablated mouse gene with a mouse containing (1) bovine or otherungulate PrP genes (which may be present in high copy numbers) or (2)chimeric mouse/ungulate PrP genes. The term hybrid includes anyoffspring of a hybrid including inbred offspring of two hybrids providedthe resulting offspring is susceptible to infection with prions withnormal infect only a genetically diverse species. A hybrid animal can beinoculated with prions and serve as a source of cells for the creationof hybridomas to make monoclonal antibodies of the invention.

The terms “susceptible to infection” and “susceptible to infection byprions” and the like are used interchangeably herein to describe atransgenic or hybrid test animal which develops a disease if inoculatedwith prions which would normally only infect a genetically diverse testanimal. The terms are used to describe a transgenic or hybrid animalsuch as a transgenic mouse Tg(MBo2M) which, without the chimeric PrPgene, would not become infected with a bovine prion but with thechimeric gene is susceptible to infection with bovine prions.

The term “ungulate” as used herein refers to any hoofed mammal. Thisincludes, but is not limited to, cows, deer, elk, sheep and goats. Forpurposes of the invention a preferred ungulate is a cow.

By “antibody” is meant an immunoglobulin protein which is capable ofbinding an antigen. Antibody as used herein is meant to include theentire antibody as well as any antibody fragments (e.g. F(ab′)₂, Fab′,Fab, Fv) capable of binding the epitope, antigen or antigenic fragmentof interest.

Antibodies of the invention are immunoreactive or immunospecific for andtherefore specifically and selectively bind to an ungulate PrP_(C)protein. Antibodies which are immunoreactive and immunospecific fornatural or native PrP^(C) are preferred. Antibodies for PrP^(C) arepreferably immunospecific—i.e., not substantially cross-reactive withrelated materials. Although the term “antibody” encompasses all types ofantibodies (e.g., monoclonal) the antibodies of the invention arepreferably produced using the phage display methodology describedherein.

By “purified antibody” is meant one which is sufficiently free of otherproteins, carbohydrates, and lipids with which it is naturallyassociated. Such an antibody “preferentially binds” to a native PrP^(C)protein, a denatured PrP^(Sc), or an antigenic fragment of each, i.e.,does not substantially recognize and bind to otherantigenically-unrelated molecules, including native PrP^(Sc). A purifiedantibody of the invention is preferably immunoreactive with andimmunospecific for a PrP^(C) protein of specific species and morepreferably immunospecific for native bovine PrP.

By “antigenic fragment” of a PrP protein is meant a portion of such aprotein which is capable of binding an antibody of the invention.

By “binds specifically” is meant high avidity and/or high affinitybinding of an antibody to a specific polypeptide i.e., epitope of a PrPprotein. Antibody binding to its epitope on this specific polypeptide ispreferably stronger than binding of the same antibody to any otherepitope, particularly those which may be present in molecules inassociation with, or in the same sample, as the specific polypeptide ofinterest e.g., binds more strongly to ungulate PrP^(C) than to otherproteins, including PrP^(C) from mammals such as humans, dogs, cats,etc. Antibodies which bind specifically to a polypeptide of interest maybe capable of binding other polypeptides at a weak, yet detectable,level (e.g., 10% or less of the binding shown to the polypeptide ofinterest). Such weak binding, or background binding, is readilydiscernible from the specific antibody binding to the compound orpolypeptide of interest, e.g. by use of appropriate controls. Ingeneral, antibodies of the invention which bind to ungulate PrP^(C) witha binding affinity of 10⁷ liters/mole or more, preferably 10⁸liters/mole or more are said to bind specifically to PrP^(C). Ingeneral, an antibody with a binding affinity of 10⁶ liters/mole or lessis not useful in that it will not bind an antigen at a detectable levelusing conventional methodology currently used.

By “detectably labeled antibody”, “detectably labeled anti-PrP” or“detectably labeled anti-PrP fragment” is meant an antibody (or antibodyfragment which retains binding specificity), having an attacheddetectable label. The detectable label is normally attached by chemicalconjugation, but where the label is a polypeptide, it couldalternatively be attached by genetic engineering techniques. Methods forproduction of detectably labeled proteins are well known in the art.Detectable labels may be selected from a variety of such labels known inthe art, but normally are radioisotopes, fluorophores, paramagneticlabels, enzymes (e.g., horseradish peroxidase), or other moieties orcompounds which either emit a detectable signal (e.g., radioactivity,fluorescence, color) or emit a detectable signal after exposure of thelabel to its substrate. Various detectable label/substrate pairs (e.g.,horseradish peroxidase/diamin-obenzidine, avidin/streptavidin,luciferase/luciferin)), methods for labeling antibodies, and methods forusing labeled antibodies are well known in the art (see, for example,Harlow and Lane, eds. (Antibodies: A Laboratory Manual (1988) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)).

The terms “treatment”, “treating” and the like are used herein togenerally mean obtaining a desired pharmacologic and/or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, particularly an ungulate,and includes:

(a) preventing the disease from occurring in a subject which may bepredisposed to the disease but has not yet been diagnosed as having it;

(b) inhibiting the disease, i.e., arresting its development; or

(c) relieving the disease, i.e., causing regression of the disease.

Abbreviations used herein include:

-   CNS for central nervous system;-   BSE for bovine spongiform encephalopathy;-   CWD for chronic wasting disease of deer or elk;-   CJD for Creutzfeldt-Jakob Disease;-   FFI for fatal familial insomnia;-   GSS for Gerstmann-Strassler-Scheinker Disease;-   Hu for human;-   HuPrP for a human prion protein;-   Mo for mouse;-   MoPrP for a mouse prion protein;-   SHa for a Syrian hamster;-   SHaPrP for a Syrian hamster prion protein;-   Bov for cow;-   BovPrP for a cow prion protein;-   Tg for transgenic;-   Tg(SHaPrP) for a transgenic mouse containing the PrP gene of a    Syrian hamster;-   Tg(HuPrP) for transgenic mice containing the complete human PrP    gene;-   Tg(ShePrP) for transgenic mice containing the complete sheep PrP    gene;-   Tg(BovPrP) for transgenic mice containing the complete cow PrP gene;-   PrP^(Sc) for the scrapie isoform of the prion protein;-   PrP^(C) for the cellular contained common, normal isoform of the    prion protein;-   MoPrP^(Sc) for the scrapie isoform of the mouse prion protein;-   MHu2M for a chimeric mouse/human PrP gene wherein a region of the    mouse PrP gene is replaced by a corresponding human sequence which    differs from mouse PrP at 9 codons;-   Tg(MHu2M) mice are transgenic mice of the invention which include    the chimeric MHu2M gene;-   MBo2M for a chimeric mouse/human PrP gene wherein a region of the    mouse PrP gene is replaced by a corresponding bovine sequence which    differs from mouse PrP at 9 codons;-   Tg(MBo2M) mice are transgenic mice of the invention which include    the chimeric MBo2M gene;-   MBo2M PrP^(C) for the scrapie isoform of the chimeric bovine/mouse    PrP gene;-   PrP^(CJD) for the CJD isoform of a PrP gene;-   Prnp^(0/0) for ablation of both alleles of an endogenous prion    protein gene, e.g., the MoPrP gene;-   Tg(SHaPrP^(+/0))81/Prnp^(0/0) for a particular line (81) of    transgenic mice expressing SHaPrP, +/0 indicates heterozygous;-   Tg(BovPrP)/Prnp^(0/0) for a hybrid mouse obtained by crossing a    mouse with a bovine prion protein gene (BovPrP) with a mouse with    both alleles of the endogenous prion protein gene disrupted;-   Tg(MBo2M)/Prnp^(0/0) for a hybrid mouse obtained by crossing a mouse    with a chimeric prion protein gene (MHu2M) with a mouse with both    alleles of the endogenous prion protein gene disrupted.

FVB for a standard inbred strain of mice often used in the production oftransgenic mice since eggs of FVB mice are relatively large and toleratemicroinjection of exogenous DNA relatively well.

General Aspects of the Invention

The present invention provides an antibody which specifically binds toan ungulate (e.g., cow, sheep or deer) PrP^(C) or denatured ungulatePrP^(Sc), but not to native ungulate PrP^(Sc). More specifically, themethods of the invention provide for the development of antibodies thatare able to recognize epitopes that are unavailable on the abnormalconformers of the prion protein, and in particular of the prion proteinfrom ungulates such as cows, sheep and deer. The antibodies anddetection methods of the invention allow the quantitativedistinguishment between the infectious and noninfectious state ofabnormal isoforms of prion protein, as well as between the abnormal andnormal isoforms of the prion protein. Preferably, the antibodies bind toa denatured ungulate PrP^(Sc) protein in situ with an affinity of 10⁷liters/mole or more, preferably 10⁸ liters/mole or more of a singlespecies. Antibodies of the invention may have an affinity for multiplespecies, e.g., multiple ungulates, or may be specific to a singlespecies, e.g., cow. The antibodies recognize an epitope of the PrP^(C)or denatured PrP^(Sc) that is unavailable in the native form ofPrP^(Sc), presumably due to the conformational difference betweenPrP^(C) and PrP^(Sc). Antibodies may be isolated, using the protocols ofthe present invention, with the ability to bind to all proteins coded bythe different mutations and/or polymorphisms of the ungulate PrP proteingene. Alternatively, a battery of antibodies (2 or more differentantibodies) can be provided wherein each antibody of the batteryspecifically binds to a protein encoded by a different mutation orpolymorphism of an ungulate PrP gene. Thus, the antibody can be bound toa support surface and used to assay a sample in vitro for the presenceof a particular allele of ungulate PrP^(C).

The antibodies of the present invention are characterized in part byisolation using a phage display library. Construction of phage displaylibraries for expression of antibodies, particularly the Fab portion ofantibodies, is well known in the art. Preferably, the phage displayantibody libraries that express antibodies are prepared according to themethods described in U.S. Pat. No. 5,223,409, issued Jun. 29, 1993 andU.S. patent application Ser. No. 07/945,515, filed Sep. 16, 1992, bothincorporated herein by reference. Procedures of the general methodologycan be adapted using the present disclosure to produce antibodies of thepresent invention.

The present invention includes a method for panning and screening ofantibodies developed against short synthetic peptides that correspond tothe hidden epitopes of BoPrP^(Sc) and in particular residues 90–120,which is designated as epitope I.

The antibodies of the present invention are especially useful to detectprions utilizing in vitro methods, in which the presence of PrP^(Sc) intissues of humans or animals indicates prion infection. Aconformation-dependent immunoassay (CDI) offers a rapid, specific, andhighly sensitive method for the detection of ungulate PrP^(Sc) using theantibodies of the invention. The assay, as the name indicates, isconformation-sensitive and can detect relatively low levels of PrP^(Sc)in brain homogenates in which PrP^(C) is present in a 100-fold excess.Prior to the present invention, rapid application of CDI for earlydetection of BSE prions in different tissues of cows was complicated bythe lack of high-affinity antibody reacting within the residues 90–120(epitope I) of the denatured bovine PrP. All the monoclonal orrecombinant antibodies generated prior to the invention have either lowaffinity for bovine PrP or recognize epitopes distant from epitope I.This epitope is critical not only for absolute detection of bovine PrP,but also for conformational sensitivity of CDI. Conformationalsensitivity of CDI is crucial for specificity of the assay and theability to distinguish PrP^(Sc) from PrP^(C). The methods of theinvention provide the rational development and specific selection ofhigh-affinity anti-PrP^(C) ungulate antibodies that can be used in,among other things, conformation-dependent immunoassays (CDI), forexample, in assays for wild type and de novo bovine, sheep, and deerprions.

A CDI assay is described in U.S. Pat. No. 5,891,641 issued Apr. 6, 1999and incorporated herein by reference in its entirety. The basic steps ofa CDI assay are shown in the flow diagram of FIG. 1. A sample which ispreferably a bovine brain sample is divided into two portions. The firstportion is contacted with an antibody of the invention which ispreferably attached to a detectable label. The level of binding to thebovine PrP^(C) is then determined. The second portion of the sample isthen treated in a manner which exposes an epitope which the antibodywill bind to, i.e. denaturing proteins within the sample. The treatmentexposes epitopes on PrP^(Sc) making it possible for the antibodies tobind the treated PrP^(Sc). Thus, if the sample had PrP^(Sc) in it, thelevel of binding to the second, treated portion will be higher ascompared to the level of binding to the first, untreated portion. Thetreatment can cause increased levels of binding to PrP^(C). Thus, someincrease is expected even when there is no PrP^(Sc) in the secondportion. This makes it necessary to adjust the level of binding on thesecond, treated portion downward some standard amount. After making thedownward adjustment, the level is compared to the level obtained withthe first portion and a determination is made as to whether PrP^(Sc) ispresent in the sample.

Using the present methods, three recombinant antibody fragments (Fabs)were isolated that bind tightly to denatured BoPrP^(Sc) but not to thenative conformation of the same protein in CDI-formatted ELISA. Allthree Fabs were generated against the 96–105 region of bovine prionprotein. Clones “O” and “S” recognized only bovine PrP, while clone “P”bound SHa, Mo, Ov, and Hu, as well as bovine PrP^(Sc). The “O” and “P”recombinant antibody fragments (Fabs) were isolated from a mouse cDNAand cloned into a vector that expresses human-mouse (HuM) chimeric Fabsin E. coli. The purified Fabs were then labeled with Europium and usedin the conformation dependent immunoassay (CDI) to measure bovine,sheep, and deer PrP^(Sc). The transgenic mice expressing bovine PrP^(Sc)will be used in the future for calibration of the CDI sensitivity withrespect of the infectious units.

The selection of antibodies and resultant assays can be performeddirectly in samples or indirectly in the brains of animals innoculatedwith a sample containing prions.

Although there are known procedures for producing antibodies from anygiven antigen, practice has shown that it is particularly difficult toproduce antibodies which bind to certain proteins e.g., PrP^(C). Thedifficulty with obtaining antibodies to PrP^(C) (and to PrP^(Sc))relates, in part, to its structural qualities. By following proceduresdescribed herein antibodies which bind ungulate PrP^(C) have beenobtained and others may follow the procedures described here to obtainother antibodies to PrP^(C) and to other proteins (e.g. PrP^(C) proteinsfrom other species) for which it is difficult to generate antibodies.

To produce antibodies of the invention it is preferable to begin withinoculating a host mammal with an innoculum from the desired ungulatePrP^(C). The host mammal may be any mammal and is preferably a hostmammal of the type defined herein such as a mouse, rat, rabbit, guineapig or hamster, and is most preferably a mouse. The host animal isinoculated with prion proteins which are endogenous to a ungulatespecies. For example a mouse is inoculated with a bovine PrP^(C)peptide. Using a normal host mammal in this manner it is possible toelicit the generation of some antibodies. However, since the host animalincludes a prion protein gene and is inoculated with PrP^(C) from agenetically diverse species, the antibodies will, if at all, only begenerated for epitopes which differ between epitopes of the prionprotein of the host animal and epitopes of the PrP^(C) from thegenetically diverse species. This substantially limits the amount ofantibodies which might be generated and decreases the ability to find anantibody which selectively binds to an ungulate PrP^(C). Thus, inattempting to generate antibodies which differentiate between prionproteins of different species it is preferable to begin the antibodyproduction process using a mammal with an intact endogenous PrP gene.

Antibodies can also be generated in animals which have an ablated prionprotein gene, i.e., a null PrP gene abbreviated as Prnp^(0/0). Thisallows antibodies to be generated against areas of an ungulate PrP^(C)that are conserved between the host animal and the ungulate PrP genes.Accordingly, the invention is also described in connection with the useof such “null” mammals and more specifically described in connectionwith “null mice.”

A null mouse can be created by inserting a segment of DNA into a normalmouse PrP gene and/or removing a portion of the gene to provide adisrupted PrP gene. The disrupted gene is injected into a mouse embryoand replaces the endogenous PrP gene via homologous recombination.

The null mouse is injected with ungulate PrP peptides to stimulate theformation of antibodies. Injections of adjuvants can be used inconjunction with the peptides to maximize the generation of antibodies.The mouse is then sacrificed and bone marrow and spleen cells areremoved. The cells are lysed, RNA is extracted and reversed transcribedto cDNA. Antibody heavy and light chains (or parts thereof) are thenamplified by PCR. The amplified cDNA library may be used as is, orfurther manipulated to create a range of variants and thereby increasethe size of the library.

An IgG phage display library is constructed by inserting the amplifiedcDNA encoding IgG heavy chain and the amplified cDNA encoding a lightchain into a phage display vector (e.g., a pComb3 vector) such that onevector contains a cDNA insert encoding a heavy chain fragment in a firstexpression cassette of the vector, and a cDNA insert encoding a lightchain fragment in a second expression cassette of the vector.

Ligated vectors are packaged by a phage display vector such asfilamentous phage M13 using methods well known in the art. The packagedlibrary is used to infect a culture of E. Coli, to amplify the number ofphage particles. After bacterial cell lysis, the phage particles areisolated and used in a panning procedure. The library created is pannedagainst a composition containing the appropriate prions. Antibodyfragments which selectively bind to PrP^(Sc) e.g., bovine PrP^(Sc) arethen isolated.

Obtaining Antibodies—Generalized Procedure

Antibodies of the invention can be obtained by a variety of techniques.One particular embodiment provides a method for generating antibodiesusing a library of proteins (i.e., antibodies or portions thereof) onthe surface of phage. The library is brought into contact with acomposition which includes PrP proteins, and in particular is anaturally occurring composition which includes PrP^(C). The phage whichbind to PrP^(C) are identified and the antibody or portion thereof whichbinds the PrP^(C) protein is isolated. It is desirable to determine thesequence of the genetic material encoding the antibody or portionthereof. Further, the sequence can be amplified and inserted, by itselfor with other genetic material, into an appropriate vector and cell linefor the production of additional antibodies. For example, a sequenceencoding a variable region which binds an epitope of PrP^(C) hidden inPrP^(Sc) can be fused with a sequence which encodes an ungulate (e.g.,bovine) constant region of an antibody to produce a constant/variableconstruct. This construct can be amplified and inserted into a suitablevector and transfected into a suitable cell line for the production ofantibodies. Procedures such as this are described within U.S. Pat. No.4,816,567, issued Mar. 28, 1989 to Cabilly, et al which is incorporatedherein by reference to disclose and describe such procedures. Further,see Bobrzecka et al. (1980) Immunology Letters, 2, pages 151–155 andKonieczny et al. (1981) Haematologia 14 (1), pages 85–91, alsoincorporated herein by reference.

When the genetic material encoding an antibody or portion thereof whichbinds a PrP^(C) protein is isolated, it is possible to use that geneticmaterial to produce other antibodies or portions thereof which have agreater affinity for binding PrP^(C) proteins. This is done by sitedirected mutagenesis technology or by random mutagenesis and selection.Specifically, individual codons or groups of codons within the sequencecan be removed or replaced with codons which encode different aminoacids. Large numbers of different sequences can be generated, amplifiedand used to express variations of the antibody or portions thereof onthe surface of additional phage. These phage can then be used to testfor the binding affinity of the antibody to PrP proteins.

The phage library can be created in a variety of different ways. Inaccordance with one procedure, a host animal such as a mouse or rat isimmunized with PrP^(C) protein. The immunization may be carried out withan adjuvant to optimize for larger amounts and types of antibodies.After allowing for sufficient time for the generation of antibodies,cells responsible for antibody production are extracted from theinoculated host mammal. RNA is isolated from the extracted cells andsubjected to reverse transcription in order to produce a cDNA library.The extracted cDNA is amplified by the use of primers and inserted intoan appropriate phage display vector. The vector allows the expression ofantibodies or portions thereof on the phage surface. It is also possibleto subject the cDNA to site directed mutagenesis prior to insertion intothe display vector. Specifically, codons can be removed or replaced withcodons expressing different amino acids in order to create a largerlibrary (i.e., a library of many variants) which is then expressed onthe surface of the phage. Thereafter, as described above, the phage arebrought into contact with the sample and phage which bind to PrP proteinare isolated.

Isolation of RNA Encoding Prion-Specific Antibodies

Combinatorial antibody library technology, e.g., antigen based selectionfrom antibody libraries expressed on the surface of M13 filamentousphage, offers a new approach to the generation of monoclonal antibodiesand possesses a number of advantages relative to hybridoma methodologieswhich are particularly pertinent to the present invention (Huse, W. D.,L. Sastry et al. (1989) Science 246:1275–1281.; Barbas, C. F., III, A.S. Fang, et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978–7982.; Burton,D. R. and C. F. Barbas, III (1994) Adv. Immunol. 57:191–280).

The present invention provides methods utilizing such technology toprovide PrP-specific monoclonal antibodies from phage antibody librariesprepared from BovPrP-immunized Prnp^(0/0) mice. The invention providesthe first monoclonal antibodies recognizing BovPrP in situ anddemonstrates the application of combinatorial libraries for cloningspecific antibodies from null mice. The present invention circumventsproblems of tolerance and more efficiently generates panels ofmonoclonal antibodies capable of recognizing diverse epitopes on Bov PrPand other PrPs in part using null mice. Prnp^(0/0) mice will develop IgGserum titers against Mo, Bov and human PrP following immunization withrelatively small quantities of purified respective PrP 27–30 inadjuvant. After allowing sufficient time to generate antibodies, theimmunized Prnp^(0/0) mice are sacrificed for hybridoma production in theconventional manner. Fusions derived from these mice secrete PrP^(C)specific antibody. The general methodologies involved in creating largecombinatorial libraries using phage display technology are described anddisclosed in U.S. Pat. No. 5,223,409 issued Jun. 29, 1993, which patentis incorporated herein by reference to disclose and describe phagedisplay methodology.

In general, the phage display anti-PrP antibody libraries are preparedby first isolating a pool of RNA that contains RNA encoding anti-PrPantibodies. To accomplish this, an animal (e.g., a mouse, rat, orhamster) is immunized with protein or peptide of interest. However,normal animals do not produce antibodies to prions at detectable orsatisfactorily high levels. This problem is avoided by immunizinganimals in which the (PrP) gene has been ablated on both alleles. Suchmice are designated Prnp^(0/0) and methods for making such mice aredisclosed in Bueler et al. (1992) Nature 356:577–582 and in WeismannPublication WO 93/10227, published May 27, 1993. Inoculation of nullanimals with PrP^(C) or a peptide of PrP^(C) results in production ofIgG serum titers against the prion (Prusiner et al. PNAS 1993). In onepreferred embodiment, the animal selected for immunization is aPrnp^(0/0) mouse described by Büeler and Weismann. Generally, the amountof protein necessary to elicit a serum antibody response in a “null”animal is from about 0.01 mg/kg to about 500 mg/kg.

The PrP protein is generally administered to the animal by injection,preferably by intravenous injection, more preferably by intraperitonealinjection. The animals are injected once, with at generally 1 to 4subsequent booster injections, preferably at least 3 booster injections.After immunization, the reactivity of the animal's antisera with theprion can be tested using standard immunological assays, such as ELISAor Western blot, according to methods well known in the art (see, forexample, Harlow and Lane, 1988, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Animalshaving prion-binding antisera may be boosted with an additionalinjection of PrP^(C).

Serum antibody levels are predictive of antibody secretion, andtherefore of levels of specific mRNA in lymphocytes, particularly plasmacells. Detection of serum antibodies, particularly relatively highlevels of serum antibodies, is thus correlated to a high level oflymphocytes such as plasma cells producing mRNA encoding those serumantibodies. Thus, plasma cells isolated from the PrP^(C) immunized micewill contain a high proportion of lymphocytes (e.g., plasma cells)producing prion-specific antibody, particularly when the plasma cellsare isolated from the mice within a short time period after the finalinjection boost (e.g., about 2 to 5 days, preferably 3 days).Immunization of the mice and the subsequent injection boosters thusserve to increase the total percentage of anti-PrP^(C)antibody-producing plasma cells present in the total population of themouse's plasma cells. Moreover, because the anti-PrP^(C) antibodies arebeing produced at or near peak serum levels, then anti-PrPantibody-producing plasma cells are producing anti-PrP^(C) antibodies,and thus mRNA encoding these antibodies at or near peak levels.

The above correlation between serum levels of antigen-specificantibodies, the number of lymphocytes producing those antigen-specificantibodies, and the amount of total mRNA encoding the antigen-specificantibodies provides a means for isolating a pool of mRNA that isenriched for the mRNA encoding antigen-specific antibodies of interest.Lymphocytes, including plasma cells are isolated from spleen and/or bonemarrow from the prion-immunized animals according to methods well knownin the art (see, for example, Huse, W. D., L. Sastry et al. (1989) (seecomments) Science 246:1275–1281). Preferably the lymphocytes areisolated about 2 to 5 days, preferably about 3 days after the finalimmunization boost. The total RNA is extracted from these cells. Methodsfor RNA isolation from mammalian cells are well known in the art (see,for example, Sambrook et al., 1989, Molecular Cloning: A LaboratoryManual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.).

Production of cDNA Encoding Antibodies from Lymphocyte mRNA

cDNA can be produced from the isolated RNA using reverse transcriptaseaccording to methods well known in the art (see, for example, Sambrooket al., supra). cDNA encoding antibody heavy chains or light chains canbe amplified using the polymerase chain reaction (PCR). The 3′ primersused to amplify heavy chain or light chain-encoding cDNAs are based uponthe known nucleotide sequences common to heavy chain or light chainantibodies of a specific antibody subclass. For example, one set ofprimers based upon the constant region of the IgG1 heavy chain-encodinggene can be used to amplify heavy chains of the IgG1 subclass, whileanother set of primers based upon the constant portion of the IgG1 lightchain-encoding gene is used to amplify the light chains of the IgG1subclass. The 5′ primers are consensus sequences based upon examinationof a large number of variable sequences in the data base. In thismanner, DNA encoding all antibodies of a specific antibody class orsubclass can be amplified regardless of antigen-specificity of theantibodies encoded by the amplified DNA. The entire gene encoding theheavy chain or the light chain can be amplified. Alternatively, only aportion of the heavy or light chain encoding gene may be amplified, withthe proviso that the product of PCR amplification encodes a heavy orlight chain gene product that can associate with its corresponding heavyor light chain and function in antigen binding, i.e., bind selectivelyto a prion protein. Preferably, the phage display product is an Fab orFv antibody fragment.

The antibody encoding cDNA selected for amplification may encode anyisotope and preferably encode a subclass of IgG. Exemplary mouse IgGsubclasses include IgG1, IgG2a, IgG2b, and IgG3. The selection of thespecific antibody subclass-encoding cDNA for amplification will varyaccording to a variety of factors, including, for example, the animal'sserum antibody response to the antigen. Preferably, the antibodysubclass-encoding cDNA selected for PCR amplification is that antibodysubclass for which the animal produced the highest titer of antibody.For example, if the titers of serum IgG1 are higher than any othersubclass of IgG detected in the serum antibody response, then cDNAencoding IgG1 is amplified from the cDNA pool.

Preferably, the heavy and light chains are amplified from the plasmacell cDNA to produce two separate amplified cDNA pools: 1) a cDNA poolcontaining heavy chain cDNA amplimer products, where the heavy chain isof a specific antibody subclass; and 2) a cDNA pool containing lightchain cDNA amplimer products, where the light chain is of a specificantibody subclass.

Antibodies from Transgenic Animals

In addition to obtaining genetic material which encodes antibodies byinfecting an animal with an antigen and thereafter extracting cells (andtheir DNA) responsible for antibody production, it is possible to obtainthe genetic material by producing a transgenic animal for producingantibodies. The described technology and transgenic animal technologycan be used to produce, e.g., chimeric mouse/bovine or fully bovineantibodies. The technology for producing chimeric or wholly foreignimmunoglobins involves obtaining from cells of transgenic animals whichhave had inserted into their germ line a genetic material encoding allor part of an immunoglobin which binds to the desired antigen. Whollybovine antibodies can be produced from transgenic mice which have hadinserted into their genome genetic material encoding bovine antibodies.Similar technology for producing such antibodies from transgenic animalsis described within PCT Publication No. WO 90/04036, published Apr. 19,1990. Further, see Goodhardt et al. (June 1987) Proc. Natl. Acad. Sci.U.S.A. 84:4229–4233, and Bucchine et al. (Mar. 26, 1987) Nature326:409–411, all of which are incorporated herein by reference todisclose and describe methods of producing antibodies from transgenicanimals.

The invention is largely described herein with respect to null micei.e., FVB mice with both alleles of the PrP gene ablated. However, otherhost animals can be used and preferred host animals are mice andhamsters, with mice being most preferred in that there existsconsiderable knowledge on the production of transgenic animals. Possiblehost animals include those belonging to a genus selected from Mus (e.g.mice) Rattus (e.g. rats) Oryctolagus (e.g. rabbits) and Mesocricetus(e.g. hamsters) and Cavia (e.g., guinea pigs). In general mammals with anormal full grown adult body weight of less than 1 kg which are easy tobreed and maintain can be used.

Vectors for use with Phage Display Antibody Libraries

The heavy chain-encoding cDNAs and the light chain-encoding cDNAs arethen preferably inserted into separate expression cassettes of anappropriate vector. Preferably the vector contains a nucleotide sequenceencoding and capable of expressing a fusion polypeptide comprising, inthe direction of amino- to carboxy-terminus, 1) a prokaryotic secretionsignal domain, 2) an insertion site for DNA encoding a heterologouspolypeptide (e.g., either the heavy or light chain-encoding cDNA) and inthe expression cassette for the heavy chain cDNA 3) a filamentous phagemembrane anchor domain.

The vector includes prokaryotic or mammalian DNA expression controlsequences for expressing the fusion polypeptide, preferably prokaryoticcontrol sequences. The DNA expression control sequences can include anyexpression signal for expressing a structural gene product, and caninclude 5′ and 3′ elements operatively linked to the expression cassettefor expression of the heterologous polypeptide. The 5′ control sequencedefines a promoter for initiating transcription, and a ribosome bindingsite operatively linked at the 5′ terminus of the upstream translatablesequence. The vector additionally includes an origin of replication formaintenance and replication in a prokaryotic cell, preferably a gramnegative cell such as E. coli. The vector can also include genes whoseexpression confers a selective advantage, such as drug resistance, to aprokaryotic or eukaryotic cell transformed with the vector.

The filamentous phage membrane anchor is preferably a domain of thecpIII or cpVIII coat protein capable of associating with the matrix of afilamentous phage particle, thereby incorporating the fusion polypeptideonto the phage surface. The secretion signal is a leader peptide domainof a protein that targets the protein. to the periplasmic membrane ofgram negative bacteria. Such leader sequences for gram negative bacteria(such as E. coli) are well known in the art (see, for example, Oliver,In Neidhard, F. C. (ed.) (1987) Escherichia coli and Salmonellatyphimurium, American Society for Microbiology, Washington, D.C.,1:56–69).

Filamentous Phage Membrane Anchors for use in the Phage Display Vector

Preferred membrane anchors for the vector are obtainable fromfilamentous phage M13, fl, fd, and equivalent filamentous phage.Preferred membrane anchor domains are found in the coat proteins encodedby gene III and gene VIII. The membrane anchor domain of a filamentousphage coat protein is a portion of the carboxy terminal region of thecoat protein, and includes a region of hydrophobic amino acid residuesfor spanning a lipid bilayer membrane and a region of charged amino acidresidues normally found at the cytoplasmic face of the membrane andextending away from the membrane. In the page fl, gene VIII coatprotein's membrane spanning region comprises the carboxy-terminal 11residues from 41 to 52 (Ohkawa et al (1981) J. Biol. Chem.256:9951–9958). An exemplary membrane anchor would consist of residues26 to 40 to cpVIII. Thus, the amino acid residue sequence of a preferredmembrane anchor domain is derived from the M13 filamentous phage geneVIII coat protein (also designated cpVIII or CP 8). Gene VIII coatprotein is present on a mature filamentous phage over the majority ofthe phage particle with typically about 2500 to 3000 copies of the coatprotein.

The amino acid residue sequence of another preferred membrane anchordomain is derived from the M13 filamentous phage gene III coat protein(also designate cpIII). Gene III coat protein is present on a maturefilamentous phage at one end of the phage particle with typically about4 to 6 copies of the coat protein. Detailed descriptions of thestructure of filamentous phage particles, their coat proteins, andparticles assembly are found in the reviews by Rached et al. (1986)Microbiol. Rev, 50:401–427 and Model et al. (1988) In: TheBacteriophages: Vol. 2, R. Calendar, ed., Plenum Publishing Co., pgs.375–456.

Preferably, the filamentous phage membrane anchor-encoding DNA isinserted 3′ of the cDNA insert in the library vector such that the phagemembrane anchor-encoding DNA can be easily excised and the vectorrelegated without disrupting the rest of the expression cassettes of thevector. Removal of the phage membrane anchor-encoding DNA from thevector, and expression of this vector in an appropriate host cell,results in the production of soluble antibody (Fab) fragments. Thesoluble Fab fragments retain the antigenicity of the phage-bound Fab,and thus can be used in assays and therapies in the manner that whole(non-fragmented) antibodies are used.

The vector for use with the present invention must be capable ofexpressing a heterodimeric receptor (such as an antibody or antibodyFab). That is, the vector must be capable of independently containingand expressing two separate cDNA inserts (e.g., the heavy chain cDNA andthe light chain cDNA). Each expression cassette can include the elementsdescribed above, except that the filamentous phage anchormembrane-encoding DNA is present only in the expression cassette for theheavy chain cDNA. Thus, when the antibody or Fab is expressed on thesurface of the phage, only the heavy chain polypeptide is anchored tothe phage surface. The light chain is not directly bound to the phagesurface, but is indirectly bound to the phage via its association withthe free portion of the heavy chain polypeptide (i.e., the portion ofthe heavy chain that is not bound to the phage surface).

Preferably, the vector contains a sequence of nucleotides that allow fordirectional ligation, i.e., a polylinker. The polylinker is a region ofthe DNA expression vector that operatively links the upstream anddownstream translatable DNA sequence for replication and transport, andprovides a site or means for directional ligation of a DNA sequence intothe vector. Typically, a directional polylinker is a sequence ofnucleotides that defines two or more restriction endonucleaserecognition sequences. Upon restriction enzyme cleavage, the two sitesyield cohesive termini to which a translatable DNA sequence can beligated to the DNA expression vector. Preferably, the two cohesivetermini are non-complementary and thereby permit directional insertionof the cDNA into the cassette. Polylinkers can. provide one or multipledirectional cloning sites, and may or may not be translated duringexpression of the inserted cDNA.

In a particular embodiment, the expression vector is capable ofmanipulating in the form of a filamentous phage particle. Such DNAexpression vectors additionally contain a nucleotide sequence thatdefines a filamentous phage origin of replication such that the vector,upon presentation of the appropriate genetic complement, can replicateas a filamentous phage in single stranded replicative form, and can bepackaged into filamentous phage particles. This feature provides theability of the DNA expression vector to be packaged into phage particlesfor subsequent isolation of individual phage particles (e.g., byinfection of and replication in isolated bacterial colonies).

A filamentous phage origin of replication is a region of the phagegenome that defines sites for initiation of replication, termination ofreplication, and packaging of the replicative form produced byreplications (see, for example, Rasched et al. (1986) Microbiol. Rev.50:401–427; Horiuchi (1986) J. Mol. Biol. 188:215–223). A preferredfilamentous phage origin of replication for use in the present inventionis an M13, fl, or fd phage origin of replication (Short et al. (1988)Nucl. Acids Res. 16:7583–7600). Preferred DNA expression vectors are theexpression vectors pCOMB8, pCKAB8, pCOMB2–8, pCOMB3, pCKAB3, pCOMB2–3,pCOMB2–3′ and pCOMB3H.

The pComb3H vector is a modified form of pComb3 in which (i) heavy andlight chains are expressed from a single Lac promoter as opposed toindividual promoters and (ii) heavy and light chains have two differentleader sequences (pg1B and ompA) as opposed to the same leader sequence(pHB). Reference for pComb3H Wang, et al (1995) J. Mol. Biol., Inpress.The principles of pComb3H are basically the same as for pComb3.

Production of the Phage Display Antibody Library

After the heavy chain and light chain cDNAs are cloned into theexpression vector, the entire library is packaged using an appropriatefilamentous phage. The phage are then used to infect a phage-susceptiblebacterial culture (such as a strain of E. coli) and the phage allowed toreplicate and lyse the cells, and the lysate isolated from the bacterialcell debris. The phage lysate contains the filamentous phage expressingon its surface the cloned heavy and light chains isolated from theimmunized animal. In general, the heavy and light chains are present onthe phage surface as Fab antibody fragments, with the heavy chain of theFab being anchored to the phage surface via the filamentous phagemembrane anchor portion of the fusion polypeptide. The light chain isassociated with the heavy chain so as to form an antigen binding site.Method of producing chimeric antibodies are described within U.S. Pat.No. 4,816,567, issued Mar. 28, 1989 to Cabilly, et al. which isincorporated herein by reference to disclose and describe suchprocedures. Further, See Bobrzecka et al. (1980) Immunology Letters, 2,pages 151–155 and Konieczny, et al (1981) Haematologia 14 (1) pages85–91 also incorporated herein by reference.

Selection of PrP^(C)-Antigen Specific Fabs from the Phage DisplayAntibody Library

Phage expressing an antibody or Fab that specifically binds a PrP^(C)epitope that is unavailable in PrP^(Sc) can be isolated using any of avariety of protocols for identification and isolation of monoclonaland/or polyclonal antibodies. Such methods include immunoaffinitypurification (e.g., binding of the phage to a columna having boundantigen) and antibody panning methods (e.g., repeated rounds of phagebinding to antigen bound to a solid support for selection of phage ofhigh binding affinity to the antigen). Preferably, the phage is selectedby panning using techniques that are well known in the art.

An exemplary panning protocol is performed in two cycles. First round ofpanning is performed against C-terminus biotinylated synthetic peptidescorresponding to the bovine residues 90–145. The peptides areimmobilized on a substrate to facilitate isolation of all theantibodies, e.g., attached to ELISA plates previously coated to highdensity with Streptavidin. Following binding of the peptides andisolation of bound clones, the selected phage are panned against aPrP^(C) protein (e.g., a native ungulate PrP^(C) or a chimericmouse/ungulate PrP^(C)). Selected Fab's are expressed in E. coli andpurified as described (Williamson, R. A., D. Peretz et al. (1996) Proc.Natl. Acad. Sci. USA 93:7279–7282; Peretz, D., R. A. Williamson, et al.(1997) J. Mol. Biol. 273:614–622).

After identification and isolation of phage expressing anti-PrP^(C)antibodies, the phage can be used to infect a bacterial culture, andsingle phage isolates identified. Each separate phage isolate can beagain screened using one or more of the methods described above. Inorder to further confirm the affinity of the phage for the antigen,and/or to determine the relative affinities of the phage for theantigen, the DNA encoding the antibodies or Fabs can be isolated fromthe phage, and the nucleotide sequence of the heavy and light chainscontained in the vector determined using methods well known in the art(see, for example, Sambrook et al., supra).

Isolation of Soluble Fabs from Phage Selected from the Phage DisplayAntibody Library

Soluble antibodies or Fabs can be produced from a modified display byexcising the DNA encoding the filamentous phage anchor membrane that isassociated with the expression cassette for the heavy chain of theantibody. Preferably, the DNA encoding the anchor membrane is flanked byconvenient restriction sites that allow excision of the anchor membranesequence without disruption of the remainder of the heavy chainexpression cassette or disruption of any other portion of the expressionvector. The modified vector without the anchor membrane sequence thenallows for production of soluble heavy chain as well as soluble lightchain following packaging and infection of bacterial cells with themodified vector.

Alternatively, where the vector contains the appropriate mammalianexpression sequences the modified vector can be used to transform aeukaryotic cell (e.g., a mammalian or yeast cell, preferably a mammaliancell (e.g., Chinese hamster ovary (CHO) cells)) for expression of theFab. Where the modified vector does not provide for eukaryoticexpression, preferably the vector allows for excision of both the heavyand light chain expression cassettes as a single DNA fragments forsubcloning into an appropriate vector. Numerous vectors for expressionof proteins in prokaryotic and/or eukaryotic cells are commerciallyavailable and/or well known in the art (see, for example Sambrook etal., supra).

Specifics of a PrP Gene and PrP Proteins

The genetic material which makes up the PrP gene is known for a numberof different species of animals (see Gabriel et al. (1992), Proc. Natl.Acad. Sci. USA 89:9097–9101). Further, there is considerable homologybetween the PrP genes in different mammals. Although there isconsiderable genetic homology with respect to PrP genes, the differencesare significant in some instances. More specifically, due to smalldifferences in the protein encoded by the PrP gene of different mammals,a prion which will infect one mammal (e.g. a human) will not normallyinfect a different mammal (e.g. a mouse). Due to this “species barrier”,it is not generally possible to use normal animals, (i.e., animal whichhave not had their genetic material related to PrP proteins manipulated)such as mice to determine whether a particular sample contains prionswhich would normally infect a different species of animal such as ahuman. The present invention provides methods for using modified,transgenic animals having ungulate PrP genes or chimeric ungulate PrPgene to detect prions in samples from ungulates. The antibodies of thepresent invention provide the means by which these ungulate prions canbe detected in assays

The major component of purified infectious prions, designated PrP 27–30,is the proteinase K resistant core of a larger native protein PrP^(Sc)which is the disease causing form of the ubiquitous cellular proteinPrP^(C). PrP^(Sc) is found only in scrapie infected cells, whereasPrP^(C) is present in both infected and uninfected cells implicatingPrP^(Sc) as the major, if not the sole, component of infectious prionparticles. Since both PrP^(C) and PrP^(Sc) are encoded by the samesingle copy gene, great effort has been directed toward unraveling themechanism by which PrP^(Sc) is derived from PrP^(C). Central to thisgoal has been the characterization of physical and chemical differencesbetween these two molecules. Properties distinguishing PrP^(Sc) fromPrP^(C) include low solubility (Meyer et al.(1986), Proc. Natl. Acad.Sci. USA 83:3693–7), poor antigenicity (Kascsak et al.(1987), “MousePolyclonal and Monoclonal Antibody to Scrapie-Associated FibrilProteins.” J. Virol. 61(12):3688–3693; Serban et al.(1990), Neurology40:110–117) protease resistance (Oesch et al.(1985), Cell 40:735–746)and polymerization of PrP 27–30 into rod-shaped aggregates which arevery similar, on the ultrastructural and histochemical levels, to thePrP amyloid plaques seen in scrapie diseased brains (Prusiner, et al(1983) Cell). By using proteinase K it is possible to denature PrP^(C)but not PrP^(Sc). To date, attempts to identify any post-transitionalchemical modifications in PrP^(C) that lead to its conversion toPrP^(Sc) have proven fruitless (Stahl, et al (1993) Biochemistry).Consequently, it has been proposed that PrP^(C) and PrP^(Sc) are in factconformational isomers of the same molecule.

Conformational description of PrP using conventional techniques has beenhindered by problems of solubility and the difficulty in producingsufficient quantities of pure protein. However, PrP^(C) and PrP^(Sc) areconformationally distinct. Theoretical calculations based upon the aminoacid sequences of PrPs from several species have predicted four putativehelical motifs in the molecule. Experimental spectroscopic data wouldindicate that in PrP^(C) these regions adopt α-helical arrangements,with virtually no β-sheet (Pan, K. M. et al (1993) PNAS 90:10962:6). Indramatic contrast, in the same study it was found that PrP^(Sc) and PrP27–30 possess significant β-sheet content, which is typical of amyloidproteins. Moreover, studies with extended synthetic peptides,corresponding to PrP amino acid residues 90–145, have demonstrated thatthese truncated molecules may be converted to either α-helical orβ-sheet structures by altering their solution conditions. The transitionof PrP^(C) to PrP^(Sc) requires the adoption of β-sheet structure byregions that were previously α-helical.

It is not entirely clear as to why antibodies of the type described inthe above cited publications will bind to PrP^(C) but not to PrP^(Sc).Without being bound to any particular theory it is suggested that suchmay take place because epitopes which are exposed when the protein is inthe PrP^(C) conformation are unexposed or partially hidden in thePrP^(Sc) configuration—where the protein is relatively insoluble andmore compactly folded together. It is pointed out that stating that anantibody binds to PrP^(C) but not to PrP^(Sc) is not correct in absoluteterms (but correct in commonly accepted terms) because some minimalbinding to PrP^(Sc) may occur. For purposes of the invention anindication that no binding occurs means that the equilibrium or affinityconstant K_(a) is 10⁶ l/mole or less. Further, binding will berecognized as existing when the K_(a) is at 10⁷ l/mole or greaterpreferably 10⁸ l/mole or greater. The binding affinity of 10⁷ l/mole ormore may be due to (1) a single monoclonal antibody (i.e., large numbersof one kind of antibodies) (2) a plurality of different monoclonalantibodies (e.g., large numbers of each of five different monoclonalantibodies) or (3) large numbers of polyclonal antibodies. It is alsopossible to use combinations or (1)–(3).

Antibody/Antigen Binding Forces

The forces which hold an antigen and antibody together are in essence nodifferent from non-specific interactions which occur between any twounrelated proteins i.e., other macromolecules such as human serumalbumin and human transferrin. These intermolecular forces may beclassified into four general areas which are (1) electrostatic; (2)hydrogen bonding; (3) hydrophobic; and (4) Van der Waals. Electrostaticforces are due to the attraction between oppositely charged ionic groupson two protein side-chains. The force of attraction (F) is inverselyproportional to the square of the distance (d) between the charges.Hydrogen bonding forces are provided by the formation of reversiblehydrogen bridges between hydrophilic groups such as —OH, —NH₂ and —COOH.These forces are largely dependent upon close positioning of twomolecules carrying these groups. Hydrophobic forces operate in the sameway that oil droplets in water merge to form a single large drop.Accordingly, non-polar, hydrophobic groups such as the side-chains onvaline, leucine and phenylalanine tend to associate in an aqueousenvironment. Lastly, Van der Waals are forces created between moleculeswhich depend on interaction between the external electron clouds.

Further information regarding each of the different types of forces canbe obtained from “Essential Immunology” edited by I. M. Roitti (6thEdition) Blackwell Scientific Publications, 1988. With respect to thepresent invention useful antibodies exhibit all of these forces. It isby obtaining an accumulation of these forces in larger amounts that itis possible to obtain an antibody which has a high degree of affinity orbinding strength to the PrP protein and in particular an antibody whichhas a high degree of binding strength to ungulate PrP^(C).

Measuring Antibody/Antigen Binding Strength

The binding affinity between an antibody and an antigen can be measuredwhich measurement is an accumulation of a measurement of all of theforces described above. Standard procedures for carrying out suchmeasurements exist and can be directly applied to measure the affinityof antibodies of the invention for PrP proteins including ungulatePrP^(C).

One standard method for measuring antibody/antigen binding affinity isthrough the use of a dialysis sac which is a container comprised of amaterial which is permeable to the antigen but impermeable to theantibody. Antigens which are bound completely or partially to antibodiesare placed within the dialysis sac in a solvent such as in water. Thesac is then placed within a larger container, which does not containantibodies or antigen but contains only the solvent e.g., the water.Since only the antigen can diffuse through the dialysis membrane theconcentration of the antigen within the dialysis sac and theconcentration of the antigen within the outer larger container willattempt to reach an equilibrium. After placing the dialysis sac into thelarger container and allowing for time to pass towards reaching anequilibrium it is possible to measure the concentration of the antigenwithin the dialysis sac and within the surrounding container and thendetermine the differences in concentration. This makes it possible tocalculate the amount of antigen which remains bound to antibody in thedialysis sac and the amount which disassociates from the antibody anddiffuses into the surrounding container. By constantly renewing thesolvent (e.g., the water) within the surrounding container so as toremove any antigen which is diffused thereinto, it is possible tototally disassociate the antibody from antigen within the dialysis sac.If the surrounding solvent is not renewed the system will reach anequilibrium and it is possible to calculate the equilibrium constant (K)of the reaction i.e., the association and disassociation between theantibody and antigen. The equilibrium constant (K) is calculated as anamount equal to the concentration of antibody bound to antigen withinthe dialysis sac divided by the concentration of free antibody combiningsites times the concentration of free antigen. The equilibrium constantor “K” value is generally measured in terms of liters per mole. The Kvalue is a measure of the difference in free energy (delta g) betweenthe antigen and antibody in the free state as compared with thecomplexed form of the antigen and antibody. When using the phage displaymethodology described below, the antibodies obtained have an affinity orK value of 10⁷ liters/mole or more.

Antibody Avidity

As indicated above the term “affinity” describes the binding of anantibody to a single antigen determinate. However, in most practicalcircumstances one is concerned with the interaction of an antibody witha multivalent antigen. The term “avidity” is used to express thisbinding. Factors which contribute to avidity are complex and include theheterogeneity of the antibodies in a given serum which are directedagainst each determinate on the antigen and the heterogeneity of thedeterminants themselves. The multivalence of most antigens leads to aninteresting “bonus” effect in which the binding of two antigen moleculesby an antibody is always greater, usually many fold greater, than thearithmetic sum of the individual antibody links. Thus, it can beunderstood that the measured avidity between an antiserum and amultivalent antigen will be somewhat greater than the affinity betweenan antibody and a single antigen determinate.

The Conformation-Dependent Assay (CDI)

The Conformation-Dependent Assay; or “CDI” allows the direct measurementof the amount of PrP^(Sc) in brain homogenates without prior digestionwith proteinase K to eliminate PrP^(C). The assay isconformation-sensitive and can detect relatively low levels of PrP^(Sc)in brain homogenates in which PrP^(C) is present in a 100-fold excess.By selective precipitation of PrP^(Sc) prior to differentialimmunoassay, PrP^(Sc) can be measured in the presence of a 3,000-foldexcess of PrP^(C). Currently, the assay can quantify less than 1 ng/mlof PrP^(Sc) in brain homogenate with a dynamic range of 5 orders ofmagnitude (Safar, J., H. Wille et al. (1998), Nat. Med,4(10):1157–1165). Since the prion titer in brain homogenates ofclinically ill CJD patients is equal to or lower than 10⁶ ID₅₀ units/mlof 5% brain homogenate (unpublished data), the differential immunoassaycan detect prion titers as low as 1 ID₅₀ unit/ml.

The CDI allows one to distinguish multiple strains of prions by plottingthe ratio of denatured/native PrP as a function of PrP^(Sc)concentration before and after limited proteinase K digestion. Incontrast, only one strain (DY) (Bessen, R. A. and R. F. Marsh (1994), J.Virol. 68:7859–7868) can be distinguished from the other seven strainsby Western blotting after limited proteolysis. Moreover, theirrelativity increased protease sensitivity of PrP^(Sc) in DY prions canlead to an underestimation of its level by immunoblotting (Scott, M. R.,D. Groth, et al. (1997), J. Virol. 71:9032–9044).

Specifically, the antibodies to ungulate residues 90–120 (epitope I)allow the CDI to detect prions in cows, deer, elk, sheep and otherungulates. The high-affinity antibody reacting within epitope I of thedenatured bovine PrP allow the CDI assay to detect, for example, thepresence of bovine prions in a test sample. This epitope is critical notonly for absolute, but also for conformational sensitivity of CDI.Conformational sensitivity of CDI is crucial for specificity of theassay and the ability to distinguish PrP^(Sc) from PrP^(C).

Pathogenic Mutations and Polymorphisms

There are a number of known pathogenic mutations in the human PrP gene.Further, there are known polymorphisms in the human, sheep and bovinePrP genes. The antibodies of the present invention may be geared torecognize specific alleles of the PrP gene. Alternatively polymorphismsor mutations known to be pathogenic in one species (e.g. human) can beadded to a peptide from an ungulate PrP. The following is a list of suchmutations and polymorphisms:

Pathogenic bovine Human Sheep Bovine mutations PolymorphismsPolymorphisms Polymorphisms 2 octarepeat insert Codon 129 Codon 171 5 or6 octarepeats Met/Val Arg/Glu 4 octarepeat insert Codon 219 Codon 136Glu/Lys Ala/Val 5 octarepeat insert 6 octarepeat insert 7 octarepeatinsert 8 octarepeat insert 9 octarepeat insert Codon 102 Pro-Leu Codon105 Pro-Leu Codon 117 Ala-Val Codon 145 Stop Codon 178 Asp-Asn Codon 180Val-Ile Codon 198 Phe-Ser Codon 200 Glu-Lys Codon 210 Val-Ile Codon 217Asn-Arg Codon 232 Met-Ala

The DNA sequence of the sheep and cow PrP genes have been determinedallowing, in each case, the prediction of the complete amino acidsequence of their respective PrP proteins. The normal amino acidsequence which occurs in the vast majority of individuals is referred toas the wild-type PrP sequence. This wild-type sequence is subject tocertain characteristic polymorphic variations. In the case of sheep PrPthe gene displays two amino acid polymorphisms at residues 171 and 136,while bovine PrP has either five or six repeats of an eight amino acidmotif sequence in the amino terminal region of the mature prion protein.While none of these polymorphisms are of themselves pathogenic, theyappear to influence prion diseases. Distinct from these normalvariations of the wild-type PrP proteins, certain mutations of the humanPrP gene which alter either specific amino acid residues of PrP or thenumber of octarepeats have been identified which segregate withinherited human prion diseases.

In order to provide further meaning to the above chart demonstrating themutations and polymorphisms, one can refer to the published sequences ofPrP genes. For example, a chicken, bovine, sheep, rat and mouse PrP geneare disclosed and published within Gabriel et al. (1992) Proc. Natl.Acad. Sci. USA 89:9097–9101. The sequence for the Syrian hamster ispublished in Basler et al. (1986) Cell 46:417–428. The PrP gene of sheepis published by Goldmann et al. (1990) Proc. Natl. Acad. Sci. USA87:2476–2480. The PrP gene sequence for bovine is published in Goldmannet al. (1991) J. Gen. Virol. 72:201–204. The sequence for chicken PrPgene is published in Harris et al. (1991) Proc. Natl. Acad. Sci. USA88:7664–7668. The PrP gene sequence for mink is published in Kretzschmaret al. (1992) J. Gen. Virol. 73:2757–2761. The human PrP gene sequenceis published in Kretzschmar et al. (1986) DNA 5:315–324. The PrP genesequence for mouse is published in Locht et al. (1986) Proc. Natl. Acad.Sci. USA 83:6372–6376. The PrP gene sequence for sheep is published inWestaway et al. (1994) Genes Dev. 8:959–969. These publications are allincorporated herein by reference to disclose and describe the PrP geneand PrP amino acid sequences.

Standardized Prion Preparation

Standardized prion preparations may be produced in order to test assaysof the invention and thereby improve the reliability of the assay.Although the preparation can be obtained from any animal it ispreferably obtained from a host animal which has brain materialcontaining prions of a test animal. For example, a transgenic mousecontaining a bovine prion protein gene can produce bovine prions and thebrain of such a mouse can be used to create a standardized bovine prionpreparation. Further, in that the preparation is to be a “standard” itis preferably obtained from a battery (e.g., 100; 1,000, or moreanimals) of substantial identical animals. For example, 100 mice allcontaining a very high copy number of bovine PrP genes (allpolymorphisms and mutations) would spontaneously develop disease and thebrain tissue from each could be combined to make a useful standardizedprion preparation. Standardized prion preparations are described anddisclosed in U.S. Pat. No. 5,908,969 issued Jun. 1, 1999 and U.S. Pat.No. 6,020,537 issued Feb. 1, 2000, both of which are incorporated hereinin their entirety.

Standardized prion preparations can be produced using any of modifiedhost mammals of the type described above. For example, standardizedprion preparations can be produced using mice, rats, rabbits, hamsters,or guinea pigs which are genetically modified so that they aresusceptible to infection with prions which prions would generally onlyinfect genetically diverse species such as a cow, sheep, deer or horseand which modified host mammals will develop clinical signs of CNSdysfunction within a period of time of 350 days or less afterinoculation with prions. The most preferred host mammal is a mouse inpart because they are inexpensive to use and because a greater amount ofexperience has been obtained with respect to production of transgenicmice than with respect to the production of other types of host animals.Details regarding making standardized prion preparation are described inU.S. Pat. Nos. 6,008,435 and 6,020,537, both of which are incorporatedherein by reference.

Once an appropriate type of host is chosen, such as a mouse, the nextstep is to choose the appropriate type of genetic manipulation to beutilized to produce a standardized prion formulation. For example, themice may be mice which are genetically modified by the insertion of achimeric gene of the invention. Within this group the mice might bemodified by including high copy numbers of the chimeric gene and/or bythe inclusion of multiple promoters in order to increase the level ofexpression of the chimeric gene. Alternatively, hybrid mice of theinvention could be used wherein mice which have the endogenous PrP geneablated are crossed with mice which have a bovine PrP gene inserted intotheir genome. There are, of course, various subcategories of such hybridmice. For example, the bovine PrP gene may be inserted in a high copynumber an/or used with multiple promoters to enhance expression. In yetanother alternative the mice could be produced by inserting multipledifferent PrP genes into the genome so as to create mice which aresusceptible to infection with a variety of different prions, i.e., whichgenerally infect two or more types of test animals. For example, a mousecould be created which included a chimeric gene including part of thesequence of a cow, a separate chimeric gene which included part of thesequence of a deer, and still another chimeric gene which included partof the sequence of a sheep. If all three different types of chimericgenes were inserted into the genome of the mouse the mouse would besusceptible to infection with prions which generally only infect a cow,deer and sheep.

After choosing the appropriate mammal (e.g., a mouse) and theappropriate mode of genetic modification (e.g., inserting a chimeric PrPgene such as MBo2M) the next step is to produce a large number of suchmammals which are substantially identical in terms of genetic materialrelated to prions. More specifically, each of the mice produced willinclude an identical chimeric gene present in the genome insubstantially the same copy number. The mice should be sufficientlyidentical genetically in terms of genetic material related to prionsthat 95% or more of the mice will develop clinical signs of CNSdysfunction within 350 days or less after inoculation and all of themice will develop such CNS dysfunction at approximately the same timee.g., within ±30 days of each other.

Once a large group e.g., 50 or more, more preferably 100 or more, stillmore preferably 500 or more of such mice are produced. The next step isto inoculate the mice with prions which generally only infect agenetically diverse mammal e.g., prions from an ungulate such as asheep, cow, deer or horse. The amounts given to different groups ofmammals could be varied. After inoculating the mammals with the prionsthe mammals are observed until the mammals exhibit symptoms of prioninfection e.g., clinical signs of CNS dysfunction. After exhibiting thesymptoms of prion infection the brain or at least a portion of the braintissue of each of the mammals is extracted. The extracted brain tissueis homogenized which provides the standardized prion preparation.

As an alternative to inoculating the group of transgenic mice withprions from a genetically diverse animal it is possible to produce micewhich spontaneously develop prion related diseases. This can be done,for example, by including extremely high copy numbers of a cow PrP geneinto a mouse genome. When the copy number is raised to, for example, 100or more copies, the mouse will spontaneously develop clinical signs ofCNS dysfunction and have, within its brain tissue, prions which arecapable of infecting humans. The brains of these animals or portions ofthe brain tissue of these animals can be extracted and homogenized toproduce a standardized prion preparation.

The standardized prion preparations can be used directly or can bediluted and titered in a manner so as to provide for a variety ofdifferent positive controls. More specifically, various known amounts ofsuch standardized preparation can be used to inoculate a first set oftransgenic control mice. A second set of substantially identical miceare inoculated with a material to be tested i.e., a material which maycontain prions. A third group of substantially identical mice are notinjected with any material. The three groups are then observed. Thethird group, should, of course not become ill in that the mice are notinjected with any material. If such mice do become ill the assay is notaccurate probably due to the result of producing mice whichspontaneously develop disease. If the first group, injected with astandardized preparation, do not become ill the assay is also inaccuratebecause the mice have not been correctly created so as to become illwhen inoculated with prions which generally only infect a geneticallydiverse mammal. However, if the first group does become ill and thethird group does not become ill the assay can be presumed to beaccurate. Thus, if the second group does not become ill the testmaterial does not contain prions and if the second group does become illthe test material does contain prions.

By using standardized prion preparations of the invention it is possibleto create extremely dilute compositions containing the prions. Forexample, a composition containing one part per million or less or evenone part per billion or less can be created. Such a composition can beused to test the sensitivity of the antibodies, assays and methods ofthe invention in detecting the presence of prions.

Prion preparations are desirable in that they will include a constantamount of prions and are extracted from an isogeneic background.Accordingly, contaminates in the preparations will be constant andcontrollable. Standardized prion preparations will be useful in thecarrying out of bioassays in order to determine the presence, if any, ofprions in various pharmaceuticals, products produced by using ungulatesincluding foods, cosmetics, etc.

Useful Applications

As indicated above and described further below in detailed examples itis possible to use the methodology of the invention to create a widerange of different antibodies. i.e., antibodies having differentspecific features. For example, antibodies can be created which bindonly to a PrP^(C) protein naturally occurring within a single ungulatespecies and not bind to a PrP^(C) protein naturally occurring withinother species. Further, the antibody can be designed so as to bind onlyto a non-infectious form of an ungulate prion protein (e.g., PrP^(C))and not bind to an infectious form (e.g., PrP^(Sc)). A single antibodyor a battery of different antibodies can then be used to create an assaydevice. Such an assay device can be prepared using conventionaltechnology known to those skilled in the art. The antibody can bepurified and isolated using known techniques and bound to a supportsurface using known procedures. The resulting surface having antibodybound thereon can be used to assay a sample in vitro to determine if thesample contains one or more types of antibodies.

The antibodies are most useful in carrying out CDI assays of the typedescribed in U.S. Pat. No. 5,891,641. In addition, the antibodies couldbe used in treatments by binding to PrP^(C) and thereby preventing itfrom converting to PrP^(Sc).

Commercial Assays

One embodiment of the invention features commercial assays allowingdetection of PrP^(Sc) in an ungulate sample by 1) digesting the samplewith an enzyme that effectively degrades PrP^(C) and which denaturesPrP^(Sc), or alternatively by successive treatment with an enzyme thatdegrades PrP^(C) (but not PrPSc) and then an enzyme which denaturesPrP^(Sc) and 2) detecting the denatured PrP^(Sc) using an antibody ofthe present invention. For example, a sample containing bovine PrPproteins (i.e., PrP^(C) and PrP^(Sc)) can be subjected to denaturationby the use of proteinase K (PK) digestion. The use of such will digestPrP^(C) but not PrP^(Sc). Following digestion with proteinase K, thesample is further digested to denature the PrP^(Sc), and the sample iscontacted with an antibody of the present invention under suitablebinding conditions. Preferably, the antibody is bound to a substrate andcan be positioned such that the sample can be easily contacted with thesubstrate material having the antibody bound thereon. If material bindsto the antibodies on the substrate the presence of infectious PrP^(Sc)is confirmed.

In another embodiment, a sample to be tested is divided into twoportions, and one is digested to denature any PrP^(Sc) in the samplewithout destroying the PrP^(C) in the sample. Both portions arecontacted with an antibody of the invention, which will bind to PrP^(C)in the untreated portion and both PrP^(C) and PrP^(Sc) in the treatedportion. Levels of PrP^(C) or PrP^(C)+PrP^(Sc) are detected and theamount of PrP^(Sc) in the sample determine from the difference indetectable signal between the two samples.

In commercial embodiments of the invention it may be desirable to useantibodies of the invention in a sandwich type assay. More particularly,the antibody of the invention may be bound to a substrate supportsurface. The denatured sample to be tested is contacted with the supportsurface under conditions which allow for binding. Thereafter, unreactedsites are blocked and the surface is contacted with a generalizedantibody which will bind to any protein thereon. The generalizedantibody is linked to a detectable label. The generalized antibody withdetectable label is allowed to bind to any denatured PrP^(Sc) bound tothe antibodies on the support surface. If binding occurs the label canbe made to become detectable such as by generating a color therebyindicating the presence of the label which indirectly indicates thepresence of PrP^(Sc) within the sample. The assay can detect denaturedPrP^(Sc) present in an amount of 1 part per million or less, even onepart per billion or less. The PrP^(Sc) may be present in a sourceselected from the group consisting of (a) a pharmaceutical formulationcontaining a therapeutically active component extracted from an animalsource, (b) food products, (c) an organ, tissue, body fluid or cellsextracted from a human source, (d) an animal-based product such asinjectables, orals, creams, suppositories, and intrapulmonary deliveryformulations, (e) a cosmetic, and (f) a pharmaceutically active compoundextracted from a mammalian cell culture.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g., amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade, and pressure is at or nearambient.

Example 1

Identification and Isolation of Anti-Bovine Antibodies

Antibodies that recognize bovine PrP^(C) or denatured PrP^(Sc) wereproduced using Prnp^(0/0) mice. Mice were immunized with syntheticbovine PrP^(C) peptide coupled to KLH and corresponding to residues96–115 of bovine PrP. Phage display libraries were constructed fromspleens from mice showing high titers of sera against the homologousantigen. Thereafter, we panned the library against synthetic peptides ofvarying length and selected over 32 different positive clones. Theselected clones were screened by CDI-formatted ELISA and specificallyevaluated by Western blot. The mouse was injected with bovine peptidesto stimulate the formation of antibodies. The mouse is then sacrificedand bone marrow and spleen cells are removed. The cells are lysed, RNAis extracted and reversed transcribed to cDNA. Antibody heavy and lightchains (or parts thereof) and then amplified by PCR. Identified lightchain sequences were isolated as follows:

Clone PELVMTQTPSSLSASLGERVSLTCRASQDIGNNLNWIQQKPDGTIKRLIYATSSLDSGVPKRFSGSRSGSDYSLTISSL(SEQ ID NO:1) ESEDFADYYCLQHDTFPLTFGGGTKLEIKRTVAAHeavy chains isolated were as follows:

                                              FR2 Clone PEVQLLEQSGAELVKPGASVKLSCTASGFNIEDSYIH    WVKQRPEQ (SEQ ID NO:2) Clone SEVQLLEQSGAELVRPGASVKLSCTASGFNIEDYYIH    WVIQRPGQ (SEQ ID NO:3) FR2                                       FR3 Clone P GLEWIGRIDPEDGETKYAPKFQG KATITADTSSNTAYLHLRRLTS (SEQ ID NO:4) Clone S GLEWIGRIDPEDGETKYAPKFQD KATLTADTSSNTAYLHLRSLTS (SEQ ID NO:5) FR3                        FR4 Clone PEDTAIYYCGR   GAYYIKEDF-  WGQGTTLTVSSASTK (SEQ ID NO:6) Clone SEDTAIYFCGR   NDGLYAGQDY  WGQGTTLTVSSASTK (SEQ ID NO:7)

An IgG phage display library was constructed by inserting an amplifiedcDNA encoding an IgG heavy chain and the amplified cDNA encoding a lightchain into a phage display vector (e.g., a pComb3 vector) such that onevector contained a cDNA insert encoding a heavy chain fragment in afirst expression cassette of the vector, and a cDNA insert encoding alight chain fragment in a second expression cassette of the vector.Ligated vectors were packaged by filamentous phage M13 using methodswell known in the art, and used to infect a culture of E. coli, so as toamplify the number of phage particles. After bacterial cell lysis, thephage particles were isolated and used in a panning procedure. Thelibrary created was panned against a composition containing bovineprions. Antibody fragments which selectively bind to the bovine PrP^(C)were then isolated. (Barbas, C. F., III and D. R. Burton (1996) TrendsBiotechnol. 14: 230–234; Williamson, R. A., D. Peretz, et al. (1996)Proc. Natl. Acad. Sci. USA 93:7279–7282.; Williamson, R. A., D. Peretzet al. (1998) J. Virol. 72:9413–9418). The epitopes of recombinant mouseFab's O, P, and S were mapped using a library of synthetic decapeptidescorresponding to the BoPrP (90–145) sequence and overlapping by 3residues. All three Fab's reacted exclusively with single linear epitopewithin residues 96–105 of bovine PrP. However, the P antibody displaybroader specificity against similar sequences in other species and thecommon epitope motive can be summarized as: HG(S,N)QWNKPSKPKTN (SEQ IDNOS: 8 and 9). This epitope is present in all ungulate PrP sequences,including bovine, mule deer, white tail deer, rod deer, elk, camel,kudu, goat, sheep, and pig. Moreover, this epitope is also present inthe sequences of PrP from other species such as ferret, cat, mink,chimp, gorilla, orangutan, presbitis, rabbit, mouse, rat, hamster,macaque, spider monkey, squirrel monkey, baboon, and marmoset.Therefore, the P clone is expected to recognize equally well all theabove listed PrP's. An antibody using clone P was isolated asEu-(HuM)Fab P, and an antibody using clone S was isolated as Eu-(HuM)FabS.

Example 2

Detection of Chimeric Bovine PrP in Mouse Brain Homogenates

The isolated antibodies Eu-(HuM)Fab P and Eu-(HuM)Fab S were tested forsensitivity using the conformation-dependent immunoassay (CDI) to detectchimeric MBo2M PrP. The chimeric recombinant protein rPrP(MBo2M) wasdiluted into 5% PrP^(0/0) mouse brain homogenate and the two bovineanti-PrP^(C) antibodies tested for their ability to detect the proteinin its native PrP^(C) form. Briefly, the brains of Prnp^(0/0) mice whichdo not express PrP protein were homogenized on ice by 3×30 sec strokesof PowerGen homogenizer (Fisher Scientific, Pittsburgh, Pa.) in PBS (pH7.4). Resulting 10% (w/v) homogenates were spun for 5 min at 500 g attable top centrifuge. The supernatant was mixed 1:1 with 4% Sarcosyl inPBS (pH 7.4). The purified recombinant PrP(MBo2M) was diluted into thehomogenate and each sample was divided in two aliquots: (1) untreatedand designated native; (2) mixed with final 4M Gdn HCI and heated for 5min at 80–100° C. and designated denatured. Both samples wore diluted20-fold by H₂O and aliquots loaded on polystyrene plate activated for 1hr with 0.2% glutaraldehyde in PBS. The plates, incubated overnight at5° C., were blocked with TBS (pH 7.8) containing 0.5% BSA (w/v) and 6%Sorbitol (w/v).

The samples were washed three time with TBS (pH 7.8) containing 0.05%(v/v) of Tween 20 and incubated for 2 hrs with Europium-labeled chimericrecombinant Fab P and S. The plates were developed after additionalwashing in enhancement solution provided by the Europium label supplier(Wallac Inc., Turku, Finland) and signal was evaluated with Discovery(Packard Inc.) time-resolved fluorescence spectroscopy. The PrPconcentration was calculated as described (Safar, J., H. Willie, et al.(1998) Nat. Med, 4(10):1157–1165) and plotted for various antibodyconcentrations (FIG. 3). The data points and bars represent averageconcentration±SEM obtained from three independent experiments at anantibody concentration 1 μg/ml. The Europium density in both recombinantantibodies is 4.3 Eu/Fab.

Example 3

Sensitivity of Detection of Bovine PrP^(sc) in Mouse Brain Homogenates

Bovine PrP^(Sc) was detected in BSE-infected Tg(BoPrP) mouse brainhomogenates using Eu-(HuM)Fab P. Samples containing serial dilutions ofBSE-infected 5% (w/v) brain homogenate in 2% Sarcosyl (w/v), prepared asdescribed in Example 2, were treated with 5 μg/ml of Proteinase K andconcentrated with 0.3% (w/v) NaPTA and 1.7 mM MgCL₂ prior to CDI.Following PTA precipitation, each sample was divided into two aliquots:(1) untreated and designated native; (2) mixed with final 4M Gdn HCt andheated for 5 min at 80–100° C. and designated denatured. Both sampleswere diluted 20-fold by H₂O and aliquots loaded on polystyrene plateactivated for 1 hr with 0.2% glutaraldehyde in PBS. The plates,incubated overnight at 5° C., were blocked with TBS (pH 7.8), containing0.5% BSA (w/v) and 6% Sorbitol (w/v). They were then washed three timeswith TBS (pH 7.8) containing 0.05% (v/v) of Tween 20 and incubated for 2hrs with Europium-labeled chimeric recombinant Fab P and S. The plateswere developed after an additional 7 washing steps in enhancementsolution provided by the Europium label supplier (Wallac Inc., Turku,Finland). The signal was evaluated with Discovery (Packard Inc.)time-resolved fluorescence spectroscopy and the PrP concentration wascalculated as described (Safar, J., H. Willie, et al. (1998) Nat. Med,4(10):1157–1165). The native and denatured aliquots from each samplewere crosslinked to glutaraldehyde-activated ELISA plates and bothaliquots were incubated with Europium labeled (HuM)Fab P antibody. Afterwashing, the signal was evaluated with Discovery (Packard Inc.)time-resolved flourescence spectroscopy. The results are expressed as aratio (FIG. 4) or difference (FIG. 5) of the signals from denatured(TRF_(D)) and native (TRF_(N)) aliquots of each sample. The dynamicrange of the detection of BoPrP^(sc) was found to be ≧100,000-fold.

Example 4

Sensitivity of Detection of Bovine PrP^(sc) in Cow Brain Homogenates

Bovine PrP^(Sc) was also detected in homogenates of BSE-infected cowsusing Eu-(HuM)Fab P. Brains of BSE-infected and normal cows werehomogenized on ice by 3×30 second strokes of PowerGen homogenizer(Fisher Scientific, Pittsburgh, Pa.) in PBS (pH 7.4). Resulting 10%(w/v) homogenates were spun for 5 min at 500 g at table top centrifuge.The supernatant was mixed 1:1 with 4% Sarcosyl in PBS (pH 7.4). The 6BSE-infected brain homogenatse were serially diluted into normal cowbrain homogenate and each aliquot was first treated with 5 μg/ml ofProteinase K for 1 hrs at 37° C. After blocking the reaction with 0.5 mMPMSF and Aprotinin and Leupeptin (2 μg/ml each), the samples wereprecipitated with NaPTA and MgCl₂ as described (Safar, J., H. Willie, etal. (1998) Nat. Med, 4(10):1157–1165) and each sample was divided intotwo aliquots: (1) untreated and designated native; (2) mixed with final4M Gdn HCI and heated for 5 min at 80–100° C. and designated denatured.Both samples were diluted 20-fold by H₂O and aliquots loaded onpolystyrene plate activated for 1 hr with 0.2% glutaraldehyde in PBS.The plates, incubated overnight at 5° C., were blocked with TBS (pH 7.8)containing 0.5% BSA (w/V) and 6% Sorbitol (w/v).

The samples were washed three times with TBS (pH 7.8) containing 0.05%(v/v) of Tween 20 and incubated for 2 hrs with Europium-labeledrecombinant chimeric Fab P. The plates were developed after additionalwashing steps in enhancement solution provided by the Europium labelsupplier (Wallac Inc., Turku, Finland). The signal was evaluated withDiscovery (Packard Inc.) time-resolved fluorescence spectroscopy and thePrP concentration was calculated as described (Safar, J., H. Willie, etal. (1998) Nat. Med, 4(10):1157–1165). Bovine PrP^(Sc) was detected inthe brain homogenates of BSE-infected British cows using Eu-(HuM) Fab P.Dynamic range of the detection of BoPRP^(Sc) is ≧10,000-fold in samplescontaining serial dilutions of BSE-infected 5% (w/v) brain homogenate in2% Sacrosyl (w/v) were treated with 5 μg/ml of Proteinase K andconcentrated with 0.3% (w/v) NaPTA and 1.7 mM MgCL₂ prior to CDI. Thenative and denatured aliquots from each sample were incubated withevaluated with Discovery (Packard Inc.) time resolved fluorescencespectroscopy from denatured (TRF_(D)) and native (TRF_(N)) aliquots ofeach sample. The results are expressed as a ratio (FIG. 6) or difference(FIG. 7) of the signals from denatured (TRF_(D)) and native (TRF_(N))aliquots of each sample.

Example 5

Strain Sensitivity of Antibody Against Bovine PrPSc in Infected CowBrain Homogenates

Difference in Eu-(HuM)Fab P detection due to differences in BSE straincharacteristics was determined using homogenates from 32 differentBritish cows infected with BSE. Brains of 32 BSE-infected cows and 7normal U.S. control cows were homogenized on ice by 3×30 sec strokes ofPowerGen homogenizer (Fisher Scientific, Pittsburgh, Pa.) in PBS (pH7.4). Resulting 10% (w/v) homogenates was spun for 5 min at 500 g attable top centrifuge. The supernatant was mixed 1:1 with 4% Sarcosyl inPBS (pH 7.4). The BSE-infected brain homogenate was serially dilutedinto uninoculated Tg(Bo) mice homogenate and each aliquot was firsttreated with 5 μg/ml of Proteinase K for 1 hrs at 37° C. After blockingthe reaction with 0.5 mM PMSF and Aprotinin and Leupeptin (2 μg/mleach), the samples were precipitated with NaPTA and MgCl₂ as described(Safar, J., H. Willie, et al. (1998) Nat. Med, 4(10):1157–1165) and eachsample was divided into two aliquots: (1) untreated and designatednative; (2) mixed with final 4M Gdn HCI and heated for 5 min at 80–100°C. and designated denatured. Both samples were diluted 20-fold by H₂Oand aliquots loaded an polystyrene plate activated for 1 hr with 0.2%glutaraldehyde in PBS. The plates, incubated overnight at 5° C., wereblocked with TBS (pH 7.8) containing 0.5% BSA (w/v) and 6% Sorbitol(w/v).

The samples were then washed three times with TBS (pH 7.8) containing0.05% (v/v) of Tween 20 and incubated for 2 hrs with Europium-labeledrecombinant chimeric Fab P. The plates were developed after additionalwashing steps in enhancement solution provided by the Europium labelsupplier (Wallac Inc., Turku, Finland). The signal was evaluated withDiscovery (Packard Inc.) time-resolved fluorescence spectroscopy and thePrP concentration was calculated as described (Safar, J., H. Willie, etal. (1998) Nat. Med, 4(10):1157–1165). Concentration of PrP 27–30plotted against denatured/native ratio determined by CDI in 32 Britishcows infected by BSE and 12 U.S. controls (FIG. 8). The data areexpressed as average±SEM. The concentration of PrP 27–30 was calculatedas described previously (Safar, J., H. Willie, et al. (1998) Nat. Med,4(10):1157–1165).

Example 6

Cross-Species Sensitivity of Eu-(HuM)Fab P

The Eu-(HuM)Fab P antibody was then tested for its ability to detectprion in a variety on ungulate species, including mule deer, elk, andwhite-tail deer. The brain homogenates of chronic wasting diseases(CWD)-infected mule deer, elk, white-tail deer, and normal controls weretreated as in Example 4 to determine the ability of Eu-(HuM)Fab Pantibody to recognize prions in these different species. The results ofCDI testing for PrP^(Sc) is shown in FIGS. 9 and 10. The results areexpressed as a ratio (FIG. 9) or difference (FIG. 10) of thetime-resolved fluorescence (TRF) signals from denatured (TRF_(D)) andnative (TRF_(N)) aliquots of each sample.

Example 7

Detection of Prions in Deer Infected with CWD

Deer PrP^(Sc) was detected in homogenates of CWD-infected deer usingEu-(HuM)Fab P. Samples containing serial dilutions of CWD-infected 5%(w/v) brain homogenate in 2% Sacrosyl (w/v) were treated with 5 μg/ml ofProteinase K and concentrated with 0.3% (w/v) NaPTA and 1.7 mM MgCL₂prior to CDI. The native and denatured aliquots from each sample werecrosslinked to glutaraldehyde-activated ELISA plate and both aliquotswere incubated with Europium labeled (HuM)Fab P antibody. After 7washing steps, the signal was evaluated with Discovery (Packard Inc.)time-resolved fluorescence spectroscopy. The results are expressed as aratio (FIG. 11) or difference (FIG. 12) of the signals from denatured(TRF_(D)) and native TRF^(N)) aliquots of each sample.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

1. A method of detecting ungulate PrP^(Sc), comprising the steps of:providing a sample suspected of containing ungulate PrP^(Sc) andungulate PrP^(C); dividing the sample into a first portion and a secondportion; contacting the first portion with a labeled antibody whichbinds to native ungulate PrP^(C) with a binding affinity of K_(a) of 10⁸l/mol or more wherein the antibody is further characterized by notbinding to PrP^(C) of a mammal other than a ungulate; treating thesecond portion in a manner in which causes any ungulate PrP^(Sc) toassume a confirmation which binds to the antibody which binds to nativeungulate PrP^(C); contacting the treated second portion with the labeledantibody; determining the level of binding of labeled antibody toPrP^(C) in the first portion; determining the level of binding oflabeled antibody to treated PrP protein in the second portion; andcomparing the level of binding of labeled antibody to PrP protein in thefirst portion with the level of binding to labeled antibody in thesecond portion and thereby determining whether the sample comprisesungulate PrP^(Sc) protein.
 2. The method as claimed in claim 1, whereinthe antibody binds to native ungulate PrP^(Sc) with a binding affinityOf K_(a) of 10⁶ l/mol or less.
 3. The method as claimed in claim 1,further comprising: homogenizing the sample prior to dividing the sampleinto a first portion and a second portion.
 4. The method as claimed inclaim 3, wherein the homogenized sample is treated with a binding agentwhich binds ungulate PrP^(Sc) in the sample.
 5. The method as claimed inclaim 4, wherein the binding agent is phosphotungstic acid (PTA).
 6. Amethod of detecting ungulate PrP^(Sc), comprising the steps of:providing a sample suspected of containing ungulate PrP^(Sc) andungulate PrP^(C); dividing the sample into a first portion and a secondportion; contacting the first portion with a labeled antibody whichbinds to native ungulate PrP^(C) with a binding affinity of K_(a) of 10⁸l/mol or more wherein the antibody is further characterized by notbinding to PrP^(C) of a mammal other than a ungulate; treating thesecond portion in a manner in which causes any ungulate PrP^(Sc) toassume a confirmation which binds to the antibody which binds to nativeungulate PrP^(C); contacting the treated second portion with the labeledantibody; determining the level of binding of labeled antibody toPrP^(C) in the first portion; determining the level of binding oflabeled antibody to treated PrP protein in the second portion; adjustingthe determined level of binding of labeled antibody to treated PrPprotein in the second portion to compensate for increasing bindingaffinity of PrP^(C) protein resulting from the treating; and comparingthe level of binding of labeled antibody to PrP protein in the firstportion with the level of binding to labeled antibody in the secondportion and thereby determining whether the sample comprises ungulatePrP^(Sc) protein.
 7. The method as claimed in claim 6, wherein theantibody binds to native ungulate PrP^(Sc) with a binding affinity OfK_(a) of 10⁶ l/mol or less.
 8. The method as claimed in claim 6, furthercomprising: homogenizing the sample prior to dividing the sample into afirst portion and a second portion.
 9. The method as claimed in claim 8,wherein the homogenized sample is treated with a binding agent whichbinds ungulate PrP^(Sc) in the sample.
 10. The method as claimed inclaim 9, wherein the binding agent is phosphotungstic acid (PTA). 11.The method of claim 1, wherein levels of binding of labeled antibody toPrP protein are determined using flow cytometry.
 12. The method of claim6, wherein levels of binding of labeled antibody to PrP protein aredetermined using flow cytometry, and the labeled antibody isfluorescence labeled.
 13. The method of claim 1, wherein the labeledantibody is Europium-labeled.
 14. The method of claim 12, wherein thelabeled antibody is Europium-labeled.
 15. The method of claim 1, whereinlevels of binding of labeled antibody to PrP protein are determinedusing time-resolved, dissociation-enhanced fluorescence.
 16. The methodof claim 6, wherein levels of binding of labeled antibody to PrP proteinare determined using time-resolved, dissociation-enhanced fluorescence.17. The method of claim 10, wherein levels of binding of labeledantibody to PrP protein are determined using time-resolved,dissociation-enhanced fluorescence.
 18. The method of claim 10, furthercomprising: adjusting the determined level of binding of labeledantibody to treated PrP protein in the second portion to compensate forincreasing the affinity of the PrP^(C) protein for the antibodyresulting from the treating.
 19. The method of claim 6, wherein theungulate is selected from the group consisting of a cow, a sheep, adeer, an elk and a horse.
 20. An assay, comprising: a support surface;and an antibody bound to the surface of the support, the antibodycharacterized by an ability to bind denatured ungulate PrP^(Sc) with abinding affinity of 10⁷ l/mole or more wherein the antibody is furthercharacterized by not binding to PrP^(C) of a mammal other than aungulate.
 21. The assay of claim 20, wherein the antibody ischaracterized by an ability to bind 50% or more denatured ungulatePrP^(Sc) in a liquid flowable sample.
 22. The assay of claim 20, whereina plurality of different antibodies are bound to the support surface andeach antibody has a K_(a) of 10⁷ l/mole or more relative to PrP^(Sc).23. A method of detecting prions in an ungulate, comprising: extractingtissue from an animal; dividing the sample into a first portion and asecond portion; contacting a the first portion with an antibodycharacterized by its ability to bind a native cow PrP^(C) in situwherein the antibody is further characterized by not binding to PrP^(C)of a mammal other than a ungulate to determine the a first PrP level;treating a second portion to denature PrP^(Sc) present in the secondportion; and contacting the denatured second sample of the tissue withthe antibody to determine a second PrP level; wherein the presence ofPrP^(Sc) in said sample is detected by subtracting the first level fromthe second level and considering an effect on the treating on the secondPrP level.
 24. The method of claim 23, wherein the tissue is braintissue and is extracted from a dead ungulate.